Liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry of ω- and (ω-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes

In order to characterize the nature of the active site of cytochrome P450 2E1, the metabolism of various fatty acids with cis/trans geometric configurations has been investigated. A system coupling atmospheric pressure chemical ionization-mass spectrometry detection with HPLC separation was developed as an alternative method for the characterization of hydroxylated metabolites of oleic and elaidic acids in rat and human liver microsomes. Oxidation of oleic and elaidic acids led to the formation of two main metabolites which were identified by LC–MS and GC–MS as ω and (ω-1)-hydroxylated (or 17-OH and 18-OH) fatty acids, on the basis of their pseudo-molecular mass and their fragmentation. The assay was accurate and reproducible, with a detection limit of 25 ng per injection, a linear range from 25 to 1128 ng per injection, no recorded interference, intra-day and inter-day precision with variation coefficients <14%. This LC–MS method was validated with oleic acid by using both radiometric and mass spectrometric detections. A significant correlation was found between the two methods in human (r=0.86 and 0.94 with P<0.05 and 0.01) and rat liver microsomes (r =0.90 and 0.85 with P<0.01 and 0.05) for 17-OH and 18-OH metabolites, respectively. HPLC coupled to mass spectrometry for the analysis of hydroxylated metabolites of elaidic acid offers considerable advantages since the method does not require use of a radioactive molecule, completely separates the two hydroxymetabolites, confirms the identification of each metabolite, and is as sensitive as the radiometric analysis method. This method allowed the comparative study of oleic and elaidic acid hydroxylations by both human and rat liver microsomal preparations.     

Biochemical and ultrastructural studies on the interaction of basic proteins with mitochondria: a primary effect on membrane configuration

The cytochrome P-450_K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D₂O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation.C₁₀-C₁₆ fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450_K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450_K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450_K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450_K     

On the rate-limiting step in W-hydroxylation of lauric acid

Incubation of lauric acid with rat liver microsomes and NADPH yielded a mixture of 11-D-, 11-L-, and 12-hydroxylauric acids. 11-L- and 11-D-hydroxylations of 11-²H₂-lauric acid were accompanied by marked isotope effects, indicating that in these reactions splitting of the CH bond is rate limiting. When 11- and 12-hydroxylations of lauric acid were carried out in D₂O, 12-hydroxylation but not 11-hydroxylation was inhibited, showing that different steps are rate limiting in the two reactions.     

Fate of a terminal olefin with Drosophila microsomes and its inhibitory effects on some P-450 dependent activities

In vitro bioassays were used to analyze the metabolism of the 11-dodecenoic acid (11-DDNA) by microsomes prepared from Drosophila melanogaster RalDDT^R strain. 11-DDNA is metabolized to 11,12-epoxylauric acid (epoxyLA) in a NADPH-dependent way. The microsomal production of epoxyLA reaches a plateau very quickly, suggesting the occurrence of an enzyme inactivation process. After incubation of microsomes with (1-¹⁴C)11-DDNA, three proteins of Mr ≈? 50 kDa were labeled. 11-DDNA inhibits the microsomal metabolism of lauric acid and 7-ethoxycoumarin in a time and NADPH-dependent process. An inhibition of metabolites generated from DDT and testosterone was also obtained but at higher concentrations. These results are discussed according to the fact that RalDDT^R is an insecticide resistant strain characterized as a high metabolizer of the insecticide DDT and also of lauric acid, testosterone, and ethoxycoumarin. © 1995 Wiley-Liss, Inc.     

Leptin and Nitric Oxide Production in Normotensive and Hypertensive Men

Objective: Recent findings have shown that leptin, the product of the obesity gene, may actively participate in the regulation of blood pressure and other cardiovascular functions through the nitric oxide (NO)‐dependent mechanism. Research Methods and Procedures: In this study, to test the hypothesis that leptin regulation of NO metabolism is impaired in hypertension, we examined the possible relationship between circulating leptin and plasma NO metabolite level in normotensive (NT) and hypertensive (HT) men. Results: There were significant correlations between circulating leptin and BMI in both the NT and HT groups (NT: r = 0.64, n = 26, p < 0.01; HT: r = 0.59, n = 22, p < 0.01). The concentration of circulating leptin was similar between the NT and HT men, although the plasma NO metabolite level (nitrite and nitrate) was significantly reduced in the HT men compared with the NT men (NT: 51.0 ± 4.9 μM, n = 26; HT: 37.1 ± 2.5 μM, n = 22, p < 0.05). The circlating leptin was significantly correlated with the plasma NO metabolite level in the overall analysis of the NT and HT men (r = 0.35, n = 48, p < 0.05). When the analysis of the correlation for the NT and HT men was performed separately, there was a significant correlation between circulating leptin and plasma NO metabolites in the NT men (r = 0.45, n = 26, p < 0.05) but not in the HT men (r = 0.15, n = 22). The results of this study are consistent with the hypothesis that leptin‐related metabolism of NO might be altered in HT men.     

Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry

This study aimed at establishing a sensitive multiple reaction monitoring‐mass spectrometry (MRM‐MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM‐MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM‐MS measurement were also determined. The method was applied to liver samples from ten non‐cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM‐based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM‐MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.     

Molecular cloning of CYP76B9, a cytochrome P450 from Petunia hybrida, catalyzing the omega-hydroxylation of capric acid and lauric acid

A cDNA encoding a cytochrome P450 (CYP76B9) was isolated from Petunia hybrida. Northern blot analysis revealed preferential expression of the gene in flowers and leaves. The recombinant yeast microsomes expressing CYP76B9 was allowed to react with capric acid and lauric acid as substrates. One major metabolite was produced from each fatty acid after incubation with yeast microsomes expressing CYP76B9. The metabolites were identified by gas chromatography-mass spectrometry (GC-MS) as omega-hydroxy capric acid and omega-hydroxy lauric acid. The kinetic parameters of the reactions were Km=9.4 microM and Vmax=13.6 mol min(-1) per mol of P450 for capric acid, and Km=5.7 microM and Vmax=19.1 mol min(-1) per mol of P450 for lauric acid. We found that the omega-hydroxy metabolites of capric acid and lauric acid can affect the plant growth of Arabidopsis thaliana. Plants grown in the presence of omega-hydroxy fatty acids exhibited shorter root length than control plants with the corresponding non-hydroxylated fatty acids.     

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the ³²P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10⁸ nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min^-1 10^-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p &lt; 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.     

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. ³H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of ³ H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that ³H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. ³H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.Abbreviations 1-AP 1-aminopyrene - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(ß-aminoethyl ether) - EM electron microscopy - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPBS HEPES-phosphate-buffered-saline - HPLC high performance liquid chromatography - NBT nitroblue tetrazolium - 1-NP 1-nitropyrene - 1-NP-4,5-diol trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene - 1-NP-9,10-diol trans-9,10-dihydro-9,10-dihydroxy-1-nitropyrene - 1-NP-4,5-oxide 1-nitropyrene-4,5-oxide - 1-NP-9,10-oxide 1-nitropyrene-9,10-oxide - 3-OH-1-NP 3-hydroxy-1-nitropyrene - 6-/8-OH-1-NP a mixture of 6- and 8-hydroxy-1-nitropyrene - PBS phosphate-buffered saline - PCB a mixture of polychlorinated biphenyls (Aroclor 1254) - TLC thin layer chromatography     

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation

The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross‐bred gilts (n = 38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute‐1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morphologies: (a) uniform spherical (n = 8); (b) heterogeneous spherical and ovoid (n = 8); (c) uniform ovoid (n = 8); (d) heterogeneous ovoid and tubular (n = 8); and (e) uniform tubular (n = 6). Uterine flushings from these pregnancies were submitted for nontargeted profiling by gas chromatography–mass spectrometry (GC–MS) and ultra performance liquid chromatography (UPLC)–MS techniques. Unsupervised multivariate principal component analysis (PCA) was performed using pcaMethods and univariate analysis of variance was performed in R with false discovery rate (FDR) adjustment. PCA analysis of the GC–MS and UPLC–MS data identified 153 and 104 metabolites, respectively. After FDR adjustment of the GC–MS and UPLC–MS data, 38 and 59 metabolites, respectively, differed (p < .05) in uterine flushings from pregnancies across the five conceptus stages. Some metabolites were greater (p < .05) in abundance for uterine flushings containing earlier stage conceptuses (i.e., spherical), such as uric acid, tryptophan, and tyrosine. In contrast, some metabolites were greater (p < .05) in abundance for uterine flushings containing later stage conceptuses (i.e., tubular), such as creatinine, serine, and urea. These data illustrate several putative metabolites that change within the uterine milieu during early porcine blastocyst elongation.     

In vitro fenoprofenyl–coenzyme a thioester formation: Interspecies variations

In vitro coenzyme A thioester formation from (?)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (?)-(R)-fenoprofen, the Lineweaver–Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar K_m values). The V_max values for the thioesterification of (?)-(R)-fenoprofen present large species variations from 2.1 ± 1.0 with sheep liver microsomes to 60.6 ± 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the V_max values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (?)-(R)-fenoprofen–CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent. © 1995 Wiley-Liss, Inc.     

Measurement of dexfenfluramine metabolism in rat liver microsomes by gas chromatography–mass spectrometry

A specific and useful method was developed for the determination of dexfenfluramine metabolism by microsomal systems utilising GC–MS. The synthesis of two metabolites 1-(3-trifluoromethylphenyl)propan-2-ol (‘alcohol') and 1-(3-trifluoromethylphenyl)-1,2-propanediol (‘diol') via straightforward routes, were confirmed by MS and NMR spectra. The conditions for extraction from alkalinised microsomal mixtures of the metabolites nordexfenfluramine, 1-(3-trifluoromethylphenyl)propan-2-one (‘ketone'), alcohol and diol, their conversion to trifluoroacetate derivatives and analysis by GC–MS–SIM are described. Calibration curves were constructed between 48 and 9662 nM and fitted to quadratic equations (r²>0.999). The method precision was good over low (121 nM) medium (2415 nM) and above medium (9662 nM) concentrations for all metabolites; the within- and day-to-day coefficients of variation ranged between 2.5–12.4% and 6.7–17.5%, respectively. The accuracy, measured as bias, was very good both within- and day-to-day (range: −0.4–12.6%, 0.8–18.9%). For most metabolites, the C.V. for the assay and bias increased at 121 nM. Dexfenfluramine metabolism by rat liver microsomes was investigated using the assay method and showed a concentration dependent increase in nordexfenfluramine and ketone metabolites over the substrate range of 5–200 μM.     

Relationship of Neck Circumference to Cardiovascular Risk Factors

Objective: To determine a relationship between neck circumference (NC) and risk factors for coronary heart disease by evaluating the components of the metabolic syndrome. Research Methods and Procedures: The study group included 561 subjects (231 men and 330 women) who had no known major medical conditions and were not receiving any medication therapy. The subjects were those who attended a family health clinic for any reason between 1998 and December 2001. Main indicators studied included NC, waist circumference, waist‐to‐hip ratio, body mass index, blood pressure, and lipoprotein, glucose, and uric acid levels. Results: Pearson's correlation coefficients indicated a significant association between NC and body mass index (men, r = 0.71; women, r = 0.81; each, p < 0.0001), waist circumference (men, r = 0.75; women, r = 0.79; each, p < 0.0001), waist‐to‐hip ratio (men, r = 0.56; women, r = 0.63; each, p < 0.0001), total cholesterol (men, r = 0.50; women, r = 0.66; each, p < 0.0001), low‐density lipoprotein‐cholesterol (men, r = 0.42; women, r = 0.60; each, p < 0.0001), triglycerides (men, r = 0.48; women, r = 0.49; each, p < 0.0001), glucose (men, r = 0.21, p < 0.001; women, r = 0.44; p < 0.0001), uric acid (men, r = 0.50, p < 0.0001; women, r = 0.60, p < 0.001), and systolic (men, r = 0.53; women, r = 0.69; each, p < 0.0001), and diastolic (men, r = 0.55; women, r = 0.65; each, p < 0.0001) blood pressure. Discussion: Higher NC is correlated positively with the factors of the metabolic syndrome; therefore, it is likely to increase the risk of coronary heart disease.     

Urinary excretion of 1-hydroxypyrene as a biomarker of exposure to polycyclic aromatic hydrocarbons from different sources

1-Hydroxypyrene (1-OHP) urinary excretion has been studied in subjects exposed to polycyclic aromatic hydrocarbons (PAH) from different sources (urban air pollution, cigarette smoking, food contamination or occupational exposure). In Study A, statistically significant differences among subjects categorized according to daily cigarette consumption were observed: 1-OHP median excretion of heavy smokers (more than 20 cigarettes per day; 1-OHP=371 ng l^-1; n=6) was significantly increased over that of non-smokers (1-OHP=160 ng l^-1; n=79), light smokers (less than 10 cigarettes per day,1-OHP=157 ng l^-1; n=7) and also medium smokers (10-20 cigarettes per day, 1-OHP=154 ng l^-1; n=13) (p&lt;0.04). In smokers, 1-OHP excretion (y, ng l^-1) increased with the intensity of cigarette consumption and was associated with self-reported number of cigarettes smoked daily (x, n) (y=20+16.6x; r=0.58, n=22, p&lt;0.01), urinary thiocyanate (x, µmoll^-1) (y=55+2.6x; r=0.57, n=20, p&lt;0.01) and cotinine (x, µg l^-1) (y=89+0.23x; r=0.62, n=17, p&lt;0.01). In Study B the influence of smoked food consumption on 1-OHP excretion was evaluated: 1-OHP excretion began to increase as soon as 3 h after a PAH-rich meal and peak values were reached between 6 and 9 h after lunch. Maximum excretion mean values were respectively 525 ng l^-1 for non-smokers (n=8) and 650 ng l^-1 for smokers (n= 4). 1-OHP concentrations in next-morning samples were back to pre-lunch levels both for non-smokers and smokers. In Study C non-smoker workers (n=28) occupationally exposed to PAH in a steel plant were investigated. At values of airborne pyrene ranging between 6 and 30 µg m^-3, excretion values of 1-OHP up to 80000 ng l^-1 were observed. The use of urinary 1-OHP as a screening test to discriminate between smokers and non-smokers in the presence of uncorrected dietary influence has been calculated according to a cut-off value of 461 ng l^-1 (reference group upper limit): the 1-OHP positive predictive value is 57%, its predictive negative value is 77%, sensitivity is 15% and specificity is 96%. In conclusion, 1-OHP appears to be a valuable biomarker of pyrene exposure. It will be nevertheless more accurate in assessing human PAH exposure from multiple sources if the influence of different kinetics for inhaled (particulate or gaseous) or ingested PAH are considered and if the role of oxidative polymorphism is adequately elucidated. The possibility of using 1-OHP to estimate the total burden of PAH from different sources or of screening groups with different PAH exposure appears to be a possible approach. However, the use of 1-OHP to evaluate the associated risk of cancer is still a premature target.     

Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K_m for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.     

Relationship of Serum Adiponectin and Leptin Concentrations with Body Fat Distribution in Humans

Objective: We investigated whether serum concentrations of adiponectin are determined by body fat distribution and compared the findings with leptin. Research Methods and Procedures: Serum concentrations of adiponectin and leptin were measured by radioimmunoassay (n = 394) and analyzed for correlation with sex, age, and body fat distribution, i.e., waist‐to‐hip ratio, waist and hip circumference, and subcutaneous adipose tissue area of the lower leg as assessed by magnetic resonance imaging. Results: After adjusting for sex and percentage of body fat, adiponectin was negatively (r = ?0.17, p < 0.001) and leptin was positively (r = 0.22, p < 0.001) correlated with waist‐to‐hip ratio. Leptin, but not adiponectin, correlated with both waist (r = 0.49, p < 0.001) and hip circumference (r = 0.46, p < 0.001). Furthermore, leptin, but not adiponectin, correlated with the proportion of subcutaneous fat of the lower leg cross‐sectional area (r = 0.37, p < 0.001). Discussion: These data suggest that both adipocytokines are associated with central body fat distribution, and serum adiponectin concentrations are determined predominantly by the visceral fat compartment.     

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively

We have separated and quantified two new minor hemoglobins named HbA_1d3a and HbA_1d3b. The level of HbA_1d3a was significantly higher in uremic than in non-uremic patients (3.00 ± 0.50% vs. 1.28 ± 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r=0.80, n=81, p<0.002) and with plasma urea concentration (r=0.78, n=81, p<0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA_1d3a is an α-chain modified by carbamylation. The HbA_1d3b level in the diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 ± 0.49 vs. 1.90 ± 0.33). This was attributed to HbA_1d3 modified by glycation. Indeed HbA_1d3b correlated significantly with HbA_1c (r=0.71, p<0.002) and with serum glucose level (r=0.62, p<0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averagd long-term uremia and glycemia in uremic and diabetic patients.     

Intracellular glutathione deficiency is associated with enhanced nuclear factor-κB activation in older noninsulin dependent diabetic patients

Diabetes mellitus may be associated with intracellular glutathione (GSH) deficiency. Since in vivo studies have shown that plasma intracellular GSH plays a key role in regulating the activation of nuclear factor κB (NF-κB), we have investigated the relationship between intracellular thiols (GSH, homocysteine, cysteine and cysteinyglycine) and NF-κB activity in the peripheral blood mononuclear cells (PBMC) of 63 elderly non-insulin dependent diabetes mellitus (NIDDM) patients (28 microalbuminurics and 35 normoalbuminurics) and 30 healthy age- and sex-matched subjects. In addition, we have measured plasma concentrations of these thiol compounds, serum concentrations of interleukin-6 (IL-6) and vascular cell adhesion molecule-1 (sVCAM-1), that are partly dependent on the NF-κB activation, as well as the serum levels of thiobarbituric acid reacting substances (TBARS), as index of lipid peroxidation. Diabetic patients with microalbuminuria (MAB) and normoalbuminuria had NF-κB activity 2.1- and 1.5-fold greater, respectively, than the control group. As compared to normoalbuminuric patients, patients with MAB had significantly higher levels of glycemia, plasma homocysteine, and serum concentrations of TBARS, IL-6 and sVCAM-1 (in all cases, p < 0.01), and significantly lower GSH content in the PBMC (p < 0.05). The intracellular GSH in PBMC correlated with NF-κB activation (r = -0.82; p < 0.0001), serum TBARS (r = -0.60; p < 0.001), and with fasting glycemia (r = -0.56; p < 0.001) in patients with MAB, whereas a weaker association between GSH levels in PBMC and NF-κB activation (r = -0.504, p < 0.001) was seen in patients without MAB. These results suggest that the decrease of intracellular GSH content in elderly NIDDM patients with MAB is strongly associated with enhanced NF-κB activation, which could contribute to the development of increased glomerular capillary permeability and its rapid progression.     

Production Rates of Cortisol in Obesity

Objective: To determine the metabolic clearance rates (MCRs) and endogenous production rates (PRs) of cortisol (F) in grades 2 and 3 obese men (n = 9) and women (n = 6). Research Methods and Procedures: The MCRs and the endogenous PRs of cortisol (F) were determined in grades 2 and 3 obese men (n = 9) and women (n = 6) using the stable isotope dilution technique and mass spectrometry. Results: In obese women, endogenous PRs of F (0.6 ± 0.4 mg/h) were similar to those of nonobese women, but MCRs of F were higher in obese women (9 ± 4 L/h) compared with nonobese women (5 + 2 L/h; p < 0.05). The MCR of F was correlated with the ratio of excreted cortisone to F metabolites. Furthermore, obese women were characterized by an increased ratio of androsterone to etiocholanolone (p < 0.01). In obese men, the MCRs (11 ± 6 L/h) and the endogenous PRs of F (0.6 ± 0.3 mg/h) were both similar to those of nonobese men, but the MCR of F was directly correlated with the ratio of excreted cortisone to F metabolites (r = 0.7833, p = 0.012). Discussion: These data demonstrate sex‐specific differences in F metabolism in obesity. The rise in MCRs of F is more pronounced in obese women than in men. However, the increase in the MCR of F is moderate in both genders and exceeds the normal range only in a subgroup of obese individuals.