SEARCH · PubMed Health

Results for “Intracellular quantification”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

PubMed1984

31P nuclear magnetic resonance studies of the fermentation of glucose to ethanol by Zymomonas mobilis.

High resolution 31P nuclear magnetic resonance spectroscopy has been employed to study the fermentation of glucose to ethanol by Zymomonas mobilis, strain ZM4, a bacterium which uses the Entner-Doudoroff pathway. The levels of nucleoside triphosphates, sugar phosphates, UDP-sugars and Pi in intact fermenting cells have been studied with a time resolution of 1 min. It is suggested that a pH gradient is established across the cell membrane during fermentation and that the intracellular pH does not rise above approximately 6.4. 31P resonances from most phosphorus-containing intermediates in the Entner-Doudoroff pathway, as well as adenosine and uridine nucleotides and a number of other intracellular metabolites, have been assigned in perchloric extracts of fermenting cells by means of a number of techniques, including two-dimensional homonuclear J-resolved and two-dimensional homonuclear shift-correlated spectroscopy. Quantification of these metabolites in spectra of extracts of fermenting cells indicates that the rate-limiting steps in the Entner-Doudoroff pathway in Z. mobilis are the conversions of glucose 6-phosphate to 6-phosphogluconate and of 3-phosphoglycerate to 2-phosphoglycerate.

Ethanol↗

PubMed1980

[Biochemical methods for the determination of a clinical protein catabolism].

1. 20 patients before surgery received enteral nutrition for three days (12 g nitrogen, 1800 Kcal). Nitrogen and urea excretions in urine during the second and third day were determined. Eleven patients had a negative nitrogen balance (-2,7 and -2,4 g/day). In these patients urea production rates were 21,1 and 20,1 g/day. An urea production rate exceeding 15 g urea/day is probable an indication for a protein catabolism. The reason for this catabolic state seems to be a decreased protein utilisation (49 and 47 percent) as the result of a metabolic stress situation. This metabolic stress was determined according the stress index (Bistrian). The patients were in a stress situation comparable to postoperative stress (+3,7 and +3,9). The determination of urea production rate and catabolic index seems a suitable tool for defining a catabolic state. 2. 3-met-histidine excretion in urine were measured in seven patients postoperatively. In different periods saline or aminoacids solutions (5% alanine) were infused. During alanine administration protein (+49%)--and 3-met-histidine excretions (+50%) increased. It is not possible to state a catabolic situation out of the 3-met-histidine excretion, because an increased excretion may result from a stimulated protein synthesis in muscle tissue or from an increased muscleprotein wasting. 3. Free amino acid pools in plasma and muscle tissue were analysed in patients with severe illness of liver and pancreas. The free amino acid pattern differed from healthy volunteers. In patients with liver disease significantly increased concentrations of phenylalanine, tyrosine and methionine were found. In patients with acute pancreatitis highly abnormal pattern of intracellular amino acids occurred with decreased concentrations of glutamine, cysteine, histidine, lysine, arginine and ornithine. The highly significant decreased concentrations of glutamine (p less than 0,01) indicate a catabolic situation of these patients. A quantification of the severity of the catabolic state out of amino acid concentrations is not possible.

Alanine↗

PubMed1989

Inflammatory cell and immune function in Merino sheep with chronic dermatophilosis.

Components of inflammatory and immunological responses were compared in 17 Merino sheep with chronic dermatophilosis (Group 1) and 15 Merino sheep that had recovered from the disease (Group 2). The functions studied included: (i) total and differential white cell counts; (ii) phagocytic function and intracellular killing by neutrophils; (iii) humoral immune response to T-dependent and T-independent antigens and to Dermatophilus congolensis. (iv) lymphocyte blastogenic responses to phytohaemagglutinin; (v) bovine serum albumen and D. congolensis antigens; (vi) quantification of T-lymphocyte subsets in skin lesions resulting after re-infection with D. congolensis zoospores. After all lesions were treated and the sheep were shorn, both groups of sheep were re-infected with D. congolensis. Both groups had similar infection rate, severity of lesions and rate of resolution after re-infection. The Group 2 sheep had significantly higher primary and secondary antibody responses to killed Brucella abortus cells than Group 1 sheep, but Group 1 sheep had higher levels of specific D. congolensis antibody throughout the trial. Neutrophils from Group 1 sheep showed a higher phagocytic rate for D. congolensis zoospores than Group 2 sheep when the zoospores were opsonised by sera from the Group 1 sheep, but there was no difference in their ability to kill ingested zoospores. Although there were some differences between the groups in the proportion of lymphocytes in lesions that reacted with monoclonal antibodies to T4, T8 and T19-19 lymphocyte markers at various times after re-infection, the sheep in Group 2 consistently had higher levels of lymphocytes reacting to a monoclonal antibody for the T6 lymphocyte antigen in skin biopsies collected 9, 15 and 21 days post-inoculation (p.i.) than did sheep in Group 1. Group 2 sheep also had higher levels of epidermal cells with immunohistochemical properties of Langerhans cells at lesion sites 15 and 21 days p.i.

Actinomycetales Infections↗

PubMed2004

Quantitative assessment of FGF regulation by cell surface heparan sulfates.

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.

Animals↗

PubMed1998

Clinical and therapeutic significance of the Na+,K+ pump*.

1. The Na+,K+-ATPase or Na+,K+-pump, mediating the active transport of Na+ and K+, which was first identified 40 years ago, is a central target for acute and long-term regulation, as well as for therapeutic intervention. Acute stimulation of the Na+,K+-pump in skeletal muscle by insulin, catecholamines, beta2-agonists or theophylline increases the intracellular uptake of K+ and accounts for the hypokalaemia elicited by these agents. Conversely, digitalis intoxication elicits hyperkalaemia via acute inhibition of the Na+, K+-pump. 2. Simple and accurate methods have been developed for the quantification of the total concentration of Na+,K+-pumps in small (0.5-5 mg) fresh or frozen biopsies of human skeletal muscle, myocardium or other tissues. This has allowed the identification of several long-term regulatory changes in the concentration of this transport system in human tissues. In skeletal muscle, upregulation is induced by training, thyroid hormones or glucocorticoids. Downregulation is seen in hypothyroidism, cardiac insufficiency, myotonic dystrophy, McArdle disease, K+ deficiency and after muscle inactivity. 3. Since the skeletal muscles contain one of the major pools of Na+,K+-pumps, these changes are important for the ability to counterregulate the hyperkalaemia elicited by exercise or the ingestion of K+. Moreover, downregulation or inhibition of the Na+, K+-pumps in skeletal muscle interferes with contractile performance. Since digitalis glycosides bind to the Na+,K+-pump, the muscles constitute a large distribution volume for these agents and are therefore an important determinant for their plasma level. 4. In cardiac insufficiency, the decrease in the concentration of Na+, K+-pumps in the myocardium is over a wide range correlated to the concomitant reduction in ejection fraction. The regulatory and pathophysiological changes in the activity and concentration of Na+, K+-pumps are important for the contractile function of skeletal muscle and heart as well as for K+ homoeostasis and the response to digitalization.

Animals↗

PubMed2003

Approaches for the quantification of protein concentration ratios.

The function of a protein is modulated by its abundance and its degree of specific post-translational modifications such as phosphorylation, glycosylation or truncation. Consequently, changes of protein concentration and the extent of their post-translational modifications has a great influence on the activity of intracellular substrate degradation processes, on the activity of intracellular biosynthetic pathways, on the cell cycle or on the function of a single cell in a whole organism. Defects in this area lead to diseases like cancer or neurodegeneration. Therefore, it is a challenge to quantify changes within the proteome in the diseased state or between developmental stages and to use the results obtained for the maximization of product yields in biotechnology or for the development of new drug targets to fight against diseases. In order to determine the intracellular concentration of a protein it is necessary to spike the cell sample with the same protein in a pure form. If the concentration changes of many proteins have to be determined, it takes a long time to obtain all these proteins in a pure form. Therefore, most approaches in this field are restricted to the determination of protein abundance ratios between two states such as diseased or healthy tissues. In this case the proteins in the sample of state A function as an internal standard for the proteins in the sample of state B and vice versa. The most common techniques in this field are the comparison of two-dimensional gel spot intensities after staining or the integration of mass spectrometric peak intensities after stable isotope labelling with (13)C, (15)N, (18)O or deuterium. The results, advantages and drawbacks of these approaches are discussed. Stable isotope labelling in combination with mass spectrometry is more accurate than the comparison of spot intensities and has the potential for the investigation of highly complex tissue samples.

Animals↗

PubMed2005

A simple model for quantification of the radiobiological effectiveness of the 10B(n,alpha)7Li capture reaction in BNCT.

A simple model has been developed for predicting radiobiological effectiveness of the neutron capture reaction in boron neutron capture therapy. This model was derived from the relationship between the cell survival from the boron capture reaction, the intracellular boron concentration, and the thermal neutron fluence. We found that the cell-killing effect of the boron capture reaction was well described using a power function of the intracellular boron concentration. Hence the relationship between cell survival from the boron capture reaction, intracellular boron concentration, and the thermal neutron fluence could be determined using a simple mathematical equation. We consider that our current approach is more appropriate and realistic than the conventional theoretical mathematical model used to estimate the radiobiological effectiveness of the neutron capture reaction in boron neutron capture therapy.

Animals↗

PubMed2002

CD2 physically associates with CD5 in rat T lymphocytes with the involvement of both extracellular and intracellular domains.

T lymphocytes can be activated and induced to proliferate through stimulation of the CD2 glycoprotein with functional combinations of CD2 antibodies. However, this mechanism of signal transduction via CD2 is still not fully understood. We have investigated which molecules on the T cell surface preferentially associate in Cis with CD2 and may regulate its signaling properties. Though a quantification method we found that CD5 represents the antigen capable of co-precipitating a larger proportion of CD2. Using co-capping assays and immunoprecipitations from cell lysates, we show that an association between CD2 and CD5 can be found in rat thymocytes, T lymphocytes and in a thymoma cell line. Possibly, this interaction is a direct one, since CD2 and CD5 transiently expressed in Cos7 cells co-precipitate each other. Furthermore, using CD2 chimeric proteins containing different domains of CD2, expressed in Cos7 cells as well as in stably transfected Jurkat cells, we show that the interaction between CD2 and CD5 is held at both the intra- and extracellular levels, but does not involve the transmembrane domain. The fact that both the extracellular and the cytoplasmic domains of CD2 interact with CD5 suggests a specific and tight association between the two molecules, possibly relevant for the fine-tuning of signal transduction in T lymphocytes.

Animals↗

PubMed1995

In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection.

We report a methodology for detecting specific DNA sequences directly inside cells, combining in situ PCR and flow cytometry. This technique is based on in situ PCR performed in the presence of digoxigenin-labeled dUTP to obtain a digoxigenin-labeled amplicon, which is then revealed by an anti-digoxigenin polyclonal antibody directly conjugated to fluorescein. Fluorescence intensity is next evaluated by flow cytometry. Our experimental models were represented by the lymphoblastoid cell lines 8E5LAV, carrying an integrated HIV-1 DNA proviral copy per cell, and A.301, infected in vitro with HIV-1 (strain IIIB). The technique is described in detail with particular attention to the optimization of critical fixation and permeabilization steps. This method allows not only the detection but also an accurate quantification of the number of positive cells in a background of negative cells. Moreover, it has the potentiality to develop into a multiparametric method for the simultaneous study of specific DNA or RNA sequences and surface or intracellular markers.

Base Sequence↗

PubMed2006

Localized sampling, electrophoresis, and biosensor analysis of Xenopus laevis cytoplasm for subcellular biochemical assays.

The Xenopus oocyte is a widely used model cell for studies of signal transduction mechanisms. Advances in microanalytical methods have made it feasible to perform rapid, localized collection of cytoplasm from individual Xenopus oocytes. Analytes contained in the cytoplasmic sample are separated by electrophoresis in a capillary and simultaneously transferred to a detection region. The development of bioengineered cells as sensitive detectors of intracellular components made quantitative measurements of native signaling molecules within the electrophoresed sample possible. Local determination of the second messenger inositol 1,4,5-trisphosphate is described to illustrate the methods for the sampling, electrophoresis, detection, and quantification of signaling molecules in single oocytes.

Animals↗

PubMed2003

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization.

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.

Amino Acid Sequence↗

PubMed1998

Assessment of the protein quality of the smooth muscle myofibrillar and connective tissue proteins of chicken gizzard.

The objective of this study was to assess the protein quality of the myofibrillar and connective tissue proteins of chicken gizzard. Protein fractions were isolated from White Leghorn chicken gizzards and quantified by detailed amino acid analysis. This quantification involved repeated extractions of ground gizzards first with Triton X-100, then with low ionic strength imidazole-buffered saline (pH 7.1), followed by either 2% SDS or by 5 M guanidine hydrochloride. The total soluble intracellular protein fraction averaged 86.3% of the total protein and the insoluble extracellular connective tissue proteins comprised the remaining 13.7%. These fractions differed significantly in their essential amino acid (EAA) profiles, with the soluble intracellular fraction having the highest percentage EAA9 (48.6 to 49.0%) and the insoluble connective tissue fraction varying from 20.8 to 23%, compared to the FAO/WHO reference pattern value of 33.9% for a 2- to 5-yr-old child. Calculated protein efficiency ratios (PER) for intracellular proteins averaged 3.02 compared with a value of 1.65 for the extracellular matrix proteins. These results provide an accurate assessment of the protein quality of smooth muscle proteins of chicken gizzard and may prove valuable for industrial control of the amount of connective tissue added to formulations of meats and poultry products.

Amino Acids↗

PubMed1994

Distribution of membrane bound guanylyl cyclases in human intestine.

The quantification and distinction of particulate guanylyl cyclases in the human intestine were considered by an enzymatic approach, which comprised the signal transduction from receptor binding to cGMP formation, and, in addition, by showing the expression of an intracellular portion of these transmembrane proteins. Basal guanylyl cyclase (GC) activities were 50 to 80 pmol cGMP formation/min/mg protein and were stimulated up to twofold by heat stable enterotoxin, but were not significantly influenced by atrial natriuretic factor. Enzymatic analysis of colonoscopic specimens pointed to the prevalence of guanylyl cyclase C in the terminal ileum and in the large bowel including colon ascendens, colon descendens, sigmoid, and rectum. The availability of sequence information on human guanylyl cyclases permitted the development of a polymerase chain reaction approach for distinguishing the expression of GC-A and GC-C in human tissue samples. The expression levels of particulate guanylyl cyclases found by polymerase chain reaction in surgical biopsy specimens confirmed the enzymatic data, in that substantial expression of GC-C was found not only in the small intestine but also in the large bowel. According to the restriction mapping of amplificates, GC-C prevailed over GC-A throughout the human intestine, particularly in the mucosal layers.

Adult↗

PubMed1995

Comparative studies of transcytosis and assembly of secretory IgA in Madin-Darby canine kidney cells expressing human polymeric Ig receptor.

Epithelial transport of polymeric IgA (pIgA) from its site of synthesis to the mucosal lumen is mediated by the polymeric Ig receptor (pIgR). During transcytosis, a disulfide bond forms between pIgR and pIgA, resulting in secretion of a covalently linked complex. To dissect further the intracellular processing and functions of pIgR, we have expressed the entire coding sequence of human pIgR cDNA in Madin-Darby canine kidney (MDCK) cells. Cloned transfected cells express human pIgR, as detected by immunofluorescence and by quantification of the cleaved extracellular domain of pIgR in culture supernatants. The function of transfected pIgR was confirmed by measuring vectorial transcytosis of 125I-labeled pIgA and its disulfide bonding to pIgR. Species specificity of transcytosis was determined by comparing transport of human, rat, and mouse pIgA in MDCK cells expressing either human or rabbit pIgR. pIgA from all three species was transported by both human and rabbit pIgR, with rat pIgA being transported to the greatest extent in each case. However, disulfide bonding was observed only with human pIgR, and was found to occur mainly inside the cell. Our results suggest that conformational differences between human and rabbit pIgR may account for differences in disulfide bonding to pIgA, and show that efficient transcytosis of pIgA is correlated better with noncovalent than covalent binding to pIgR.

Animals↗

PubMed2004

Quantification of thioredoxin mRNA expression in the rat hippocampus by real-time PCR following oxidative stress.

Thioredoxin (Trx) is a multifunctional protein with a redox-active disulfide/dithiol in the active site. Thioredoxin, with its redox-regulating and reactive oxygen species (ROS) scavenging activities, plays several important biologic roles both in intracellular and extracellular compartments. The purpose of this report was to quantify the relative expression of Trx in rat hippocampus following an oxidative stress-involving treatment such as kainic acid (KA) using real-time PCR and the 2(-DeltaDeltaC(T)) method. The relative changes in expression of Trx mRNA in KA-treated and control animals were significantly different as 2.02 +/- 0.77 and 1.0 +/- 0.26, respectively (P<0.05). Minimum and maximum n-fold changes in Trx expression in KA-treated and control animals were determined as (1.4-5.2) and (0.8-1.3), respectively. Thus, real-time PCR and the 2(-DeltaDeltaC(T)) method for data analysis from real-time PCR were found to be an accurate and sensitive method for quantifying Trx mRNA levels.

Animals↗

PubMed2002

Mouse resident peritoneal macrophages partially control in vitro infection with Coxiella burnetii phase II.

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is a small, dimorphic, obligate intracellular bacterium, sheltered within large, acidified, and hydrolase-rich phagosomes. Although several primary and established cell lines, macrophage-like cells, and primary macrophages from other species have been infected with C. burnetii, the infection of mouse primary macrophages has not been sufficiently characterized. In this report quantification of DAPI (4', 6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy, and transmission electron microscopy were used to compare the infection of three mouse-derived cells, L929 fibroblasts, J774 macrophage-like cells, and resident peritoneal macrophages, with a phase II clone of C. burnetii known to be non-virulent for mammals. Infected peritoneal phagocytes differed from L929 or J774 cells in that: (a) large vacuoles took longer to appear (3-5 d instead of 2), and were only found in a subset (20-30%) of macrophages, as opposed to in more than 70% of the other cells; (b) total and vacuole-associated relative bacterial loads in L929 and J774 cells were several-fold higher than in peritoneal macrophages; (c) estimated doubling times of the bacteria were about 68 h in the primary macrophages, 18 h in J774 and 22 h in L929 cells. Thus, mouse resident peritoneal macrophages control both the formation of the large vacuoles and the intracellular proliferation of C. burnetii phase II.

Animals↗

PubMed2006

Quantitative immunoassay to measure plasma and intracellular atazanavir levels: analysis of drug accumulation in cultured T cells.

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 microl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Quantification of menstrual and diurnal periodicities in rates of cholesterol and fat synthesis in humans.

The mass isotopomer distribution analysis (MIDA) technique is applied here in men and menstruating women to quantify periodicities in the biosynthesis of serum cholesterol and very low density lipoprotein (VLDL)-palmitate. The isotopic enrichment of the true biosynthetic precursor (intracellular acetyl-CoA) during oral or intravenous administration of sodium[1-13C]- or [2-13C]acetate was calculated from mass isotopomer fractional abundances in free cholesterol and VLDL-palmitate, determined by gas chromatography-mass spectrometry (GC-MS). To convert fractional into absolute cholesterol synthesis rates, decay rate constants of plasma cholesterol were determined from the die-away curves of endogenously labeled high-mass isotopomers. Oral [13C]acetate was a 3-4 times more efficient means of labeling the precursor pool for VLDL-palmitate than was intravenous [13C]acetate, consistent with a splanchnic site of VLDL-fatty acid synthesis, whereas the precursor for free cholesterol had an intermediate enrichment, suggesting a contribution from extra-splanchnic tissues as well. Endogenous synthesis of serum cholesterol was 8-11 mg/kg per day (an estimated 65-75% of input into serum cholesterol); it was 1.5- to 3-fold higher at night than during the day (37-49 mg/h at night compared to 9-23 mg/h during the day) and did not vary over the menstrual cycle (608-697 mg/day). In contrast, endogenous synthesis of fatty acids made a relatively minor contribution to body fat pools (1/10-1/20) of input into VLDL-palmitate) compared to dietary fat intake; it was greater in the day-time, and was influenced by menstrual cycle (3-fold elevated in the follicular phase compared to the luteal phase), and body composition (higher in obese men than normal weight men, r2 = 0.59 for lipogenesis vs. body mass index). Factors responsible for periodicities in endogenous lipid synthesis can be studied in humans using this approach.

Acetates↗