Role of cholesterol synthesis in regulation of bile acid synthesis and biliary cholesterol secretion in humans

We used lovastatin, a specific inhibitor of HMG-CoA reductase, to study the role of cholesterol synthesis in regulation of both bile acid synthesis, measured by release of 14CO2 from [26-14C]cholesterol, and biliary cholesterol secretion, measured by standard marked perfusion techniques, in humans. Six volunteers were studied in each of four periods: a) control; b) 6-10 hours after a single 40 mg oral dose of lovastatin to study acute effects; c) after 5-6 weeks of lovastatin 40 mg orally twice a day to study steady-state effects; and d) 24 h after cessation of chronic lovastatin. Mean bile acid synthesis fell to 69% of control (P less than 0.01) after single-dose lovastatin and remained at 83% of control after 5-6 weeks on lovastatin (P less than 0.05). After withdrawal of lovastatin, mean bile acid synthesis was 88% of control (NS). Mean biliary cholesterol secretion did not change after single-dose lovastatin (103% of control), but fell to 81% of control during chronic lovastatin treatment (P less than 0.05). After withdrawal of lovastatin, mean cholesterol secretion remained at 80% of control (P less than 0.05). These data suggest that in humans cholesterol synthesis is an immediate regulator of bile acid synthesis. Cholesterol synthesis also regulates biliary cholesterol secretion, but the effect is not immediate and therefore may be indirect.     

Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of ²H from orally supplied ²H₂O into stable C-H bonds on the tripeptide. The incorporation of ²H₂O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with ²H₂O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N = 6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with ²H₂O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of ²H₂O and a statistical basis for estimating N.     

Facile synthesis of deuterated estrogens

The acid catalyzed exchange of the aromatic protons, ortho to the phenolic hydroxyl moiety in estriol, estradiol, and estrone is described. The acidic protons adjacent to the keto function in estrone are also exchanged leading to tetradeuterioestrone (80.0 atom%). Estriol was converted to dideuterioestriol (81.2 atom%), and estradiol into dideuterioestradiol (90.5 atom%).     

Serum lathosterol concentration is an indicator of whole-body cholesterol synthesis in humans

The power of serum lathosterol concentration as an indicator of whole-body cholesterol synthesis was investigated in 47 human volunteers consuming two diets differing in fatty acid composition. The cholesterol balance (fecal excretion of neutral and acid steroids minus cholesterol intake) was strongly correlated with the serum level of total (free plus esterified) lathosterol and also with the ratio of serum lathosterol over serum cholesterol, both on a diet rich in polyunsaturated fatty acids (r = 0.74 for the ratio) and one containing mainly saturated fatty acids (r = 0.70 for the ratio). In a subgroup for which the serum levels of free lanosterol and other free methylsterols were also quantitated, the correlations of these levels (expressed relative to serum free cholesterol) with the cholesterol balance were lower than that found for total lathosterol (expressed relative to serum total cholesterol). A further corroboration was obtained by measuring the lathosterol/cholesterol ratio in 20 patients with familial hypercholesterolemia before and during treatment with the hydroxymethylglutaryl coenzyme A reductase inhibitor Mk-733. The ratio was lowered by 47% during drug treatment, suggesting a significant decrease of the cholesterol balance in these patients. We conclude, from the various indicators proposed to monitor whole-body cholesterol synthesis, that the lathosterol/cholesterol ratio in serum appears preferable with respect to indicative power and ease of quantitation.     

Bioavailability of synthetic and biosynthetic deuterated lycopene in humans

Current knowledge of the bioavailability of lycopene in humans is limited due to the inability to distinguish newly administered lycopene from the body reserves of lycopene. A quantitative method to assess the absorption and relative bioavailability of newly absorbed synthetic or natural lycopene was developed using two deuterated lycopene sources, in conjunction with an advanced LC/APCI-MS (liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry) to analyze newly absorbed lycopene in blood samples of study subjects. Two subjects (1 male and 1 female) consumed hydroponically grown tomatoes containing deuterium-enriched lycopene (80-84 g wet weight tomato containing 16.3 and 17.4 micromol lycopene, respectively) and two subjects (1 male, and 1 female) consumed 11 micromol synthetic (2)H(10) lycopene in 6 g of corn oil. Tomatoes were steamed and pureed. The doses were given together with a liquid formulated drink with 25% energy from fat. Our results showed that up to 34 days after taking an oral (2)H(10) lycopene dose (synthetic or from tomato) with a liquid formula drink, the area under the curve of the average serum percent enrichment response of synthetic lycopene reached 33.9 (+/-1.7) nmol-day/micromol lycopene in the dose, whereas that of lycopene from the tomato dose was 11.8 (+/-0.3) nmol-day/mumol lycopene in the dose. Our study provides evidence that the absorption of physiological levels of lycopene in intrinsically labeled tomatoes can be studied in humans. From these preliminary investigations, we find that the bioavailability of synthetic lycopene in oil appears to be about three times higher than that of lycopene from steamed and pureed tomatoes.     

Measurements of fatty acid synthesis by incorporation of deuterium from deuterated water.

Fatty acid synthesis by perfused livers of rat is measured by using D2O as tracer. The newly synthesized, deuterium-labeled fatty acids are separated from unlabeled fatty acids by gas chromatography using glass capillary columns. The areas of the deuterium-labeled peaks are proportional to the amounts of fatty acids synthesized. The absolute amounts of the individual fatty acids synthesized are obtained by use of an internal standard. The number of deuterium atoms incorporated, as determined by mass spectrometry, is proportional to the D2O concentration of the perfusate, except at very high concentrations of D2O. The relative retention times of the newly synthesized, deuterium-labeled fatty acids are proportional to their deuterium content.     

Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological activities. Identification and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly facilitated by the availability of authentic standards, and deuterated and fluorinated analogs are valuable as internal standards for quantitation. We describe the preparation, purification and characterization of 43 oxygenated sterols, including the 4 beta-hydroxy, 7 alpha-hydroxy, 7 beta-hydroxy, 7-keto, and 19-hydroxy derivatives of cholesterol and their analogs with 25,26,26,26,27,27,27-heptafluoro (F7) and 26,26,26,27,27,27-hexadeuterio (d6) substitution. The 7 alpha-hydroxy, 7 beta-hydroxy, and 7-keto derivatives of (25R)-cholest-5-ene-3 beta, 26-diol (1d) and their 16,16-dideuterio analogs were also prepared. These d2-26-hydroxysterols and [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol (1e) were synthesized from [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol diacetate (2e), which can be prepared from diosgenin. The highly specific deuterium incorporation at C-16 in 1e and 2e should be useful in mass spectral analysis of 26-hydroxycholesterol samples by isotope dilution methods. The delta 5-3 beta, 7 alpha, 26- and delta 5-3 beta, 7 beta, 26-triols were regioselectively oxidized/isomerized to the corresponding delta 4-3-ketosteroids with cholesterol oxidase. Also described are 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, its 5 beta,6 beta-isomer, cholestane-3 beta, 5 alpha,6 beta-triol, their F7 and d6 derivatives, and d3-25-hydroxycholesterol, which was prepared from 3 beta-acetoxy-27-norcholest-5-en-25-one (30). The 43 oxysterols and most synthetic intermediates were isolated in high purity and characterized by chromatographic and spectroscopic methods, including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Detailed mass spectral assignments are presented, and 1H NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols and for the side-chain protons of 30.     

Measurement of rates of cholesterol synthesis using tritiated water

Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14C-labeled substrates or [3H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [3H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3H are incorporated into cholesterol from [3H]water in different tissues of several animal species, so that the 3H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [3H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [3H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo.     

Use of the doubly labeled water method for measurement of energy expenditure, total body water, water intake, and metabolizable energy intake in humans and small animals

The basis of the doubly labeled water method is measurement of the differential rates of disappearance of two isotopes of water (H2 18O and either 2H2O or 3H2O, administered at the start of the study) from body water. Published studies indicate that, in its current forms, this technique can be used to provide accurate and reasonably precise information on carbon dioxide production, total body water, and water intake in free-living humans and many small animals. Total energy expenditure can be calculated from carbon dioxide production with little loss of precision. Metabolizable energy intake can also be predicted, as the sum of total energy expenditure plus an estimate for the change in body energy stores during the measurement, but this prediction is unlikely to be accurate and precise unless the subject is in approximate energy balance.     

Use of sulfate production as a measure of short-term sulfur amino acid catabolism in humans

There is no fully satisfactory method for measuring amino acid catabolism in the nonsteady state that follows normal protein consumption. Because sulfate is the major product of sulfur amino acid catabolism, we tested whether its production can be accurately depicted using simple tracer or nontracer approaches under basal conditions and after the intravenous administration of a known amount of sulfate. In the basal postabsorptive state, serum sulfate concentration and urinary sulfate excretion remained constant for many hours, but the apparent steady-state serum sulfate rate of appearance achieved with primed continuous oral administration of sodium [(34)S]sulfate was 20% higher than urinary sulfate excretion. By contrast, after magnesium sulfate infusion, the increase in sulfate production above basal accounted for 95% over 6 h and 98% over 9 h of the administered dose when measured simply as urinary inorganic sulfate excretion corrected for changes in its extracellular fluid content. Using the latter method, we measured sulfate production after oral methionine and intravenous infusion of methionine in a mixture of other essential amino acids. Sulfate production above basal accounted for 59% over 6 h and 75% over 9 h of the oral methionine dose. Similar results were obtained with the mixed amino acid infusion, but interpretation of the latter experiment was limited by the mild protein sparing (and, hence, reduced endogenous sulfate production) induced by the amino acid infusion. We conclude that a simple nontracer method can provide an accurate measure of sulfate production and, hence, sulfur amino acid catabolism over collection periods as short as 6 h after a test meal. A significant portion of the sulfur derived from methionine appears to be retained in nonprotein compounds immediately after its ingestion.     

Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans

Subjects with high plasma cholesterol levels exhibit a high production of VLDL apolipoprotein B-100 (apoB-100), suggesting that cholesterol is a mediator for VLDL production. The objective of the study was to examine whether endogenous cholesterol synthesis, reflected by the lathosterol-cholesterol ratio (L-C ratio), affects the secretory rates of different VLDL subfractions. Ten healthy subjects were studied after overnight fasting. During a 10 h primed, constant infusion of 13C-valine (15 micromol/kg/h), enrichment was determined in apoB-100 from ultracentrifugally isolated VLDL-1 and VLDL-2 by gas chromatography mass spectrometry. The synthesis rates of VLDL-1 apoB-100 and VLDL-2 apoB-100, catabolism, and transfer were estimated by compartmental analysis. Mean VLDL-1 apoB-100 pool size was 90 +/- 15 mg, and mean VLDL-2 apoB-100 pool size was 111 +/- 14 mg. Absolute synthesis rate of VLDL-1 apoB-100 was 649 +/- 127 mg/day and 353 +/- 59 mg/day for VLDL-2 apoB-100. There was a strong association between the absolute synthesis rate of VLDL-2 apoB-100 and L-C ratio (r 2 = 0.61, P < 0.01). In contrast, no correlation was observed between L-C ratio and absolute synthesis rate of VLDL-1 apoB-100 (r 2 = 0.302, P = 0.09). In conclusion, these data provide additional support for an independent regulation of VLDL-1 apoB-100 and VLDL-2 apoB-100 production.Endogenous cholesterol synthesis is correlated only with the VLDL-2 apoB-100 production.     

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum.

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.     

Enzymatic ester synthesis with continuous measurement of water activity

Ester synthesis from aliphatic monoalcohols and organic acids was investigated by using a microbial lipase. The reaction medium only contained the substrates and the enzyme without addition of water or organic solvent. During the reaction, water was produced and the water activity (aw) increased. Batch reactors and continuous-flow reactors were used. In batch, the aw was 0.13 at the beginning of the reaction and increased to reach a plateau at 0.77, after which ester synthesis continued without modification of the aw. Different alcohols and acids were tried in solid-liquid reactors, and all cases synthesis occurred, leading to a significant increase in the water activity. For continuous-flow reactors, the use of silica beads retaining water inside the reactor where the enzymatic reaction took place resulted in some control of the enzymatic reaction by changing the aw.     

Chemical synthesis of deuterated folate monoglutamate and in vivo assessment of urinary excretion of deuterated folates in man

the synthesis and in vivo application of stable-isotopically labeled folic acid was investigated to devise methods suitable for studies of folate metabolism in human subjects. Glutamate-labeled tetradeutero-pteroylglutamic acid (d4-folic acid) was prepared by mixed anhydride coupling of N10-trifluoroacetylpteroic acid and dimethyl L-[3,3,4,4-2H4]glutamic acid, saponification in sodium deuteroxide, and chromatographic purification. Retention of the isotopic label was verified by proton NMR and mass spectrometry of the para-aminobenzoylglutamic acid product of C9-N10 bond cleavage. A method was devised for determination of of isotopic enrichment of urinary d4-folates derived from orally administered d4-folic acid using affinity chromatographic purification, chemical cleavage of the C9-N10 bond, HPLC isolation of the p-[2H4]aminobenzoylglutamate product, followed by negative-ion chemical-ionization gas chromatography/mass spectrometry. Data concerning the urinary excretion of d4-folates derived from an oral dose of d4-folic acid in an adult human are presented.     

Use of the doubly labeled water technique in humans during heavy sustained exercise

We measured energy expenditure with the doubly labeled water technique during heavy sustained exercise in the Tour de France, a bicycle race lasting more than 3 wk. Four subjects were observed for consecutive intervals of 7, 8, and 7 days. Each interval started with an oral isotope dose to reach an excess isotope level of 200 ppm 18O and 130 ppm 2H. The biological half-lives of the isotopes were between 2.25 and 3.80 days. Energy expenditure was compared with simultaneous measurements of energy intake, and body mass and body composition did not change significantly. The doubly labeled water technique gave higher values for energy expenditure than the food record technique. The discrepancy showed a systematic increment from the first to the third interval, being 12.9 +/- 7.9, 21.4 +/- 9.8, and 35.3 +/- 4.4% of the energy expenditure calculated from dietary intake, respectively. Possible explanations for the discrepancy are discussed. The subjects reached an average daily metabolic rate of 3.4-3.9 or 4.3-5.3 times basal metabolic rate based on the food record technique and the doubly labeled water technique, respectively. Thus, when measured with the same technique, the energetic ceiling for performance in humans is comparable with that of animals like birds.