Localization of atherosclerosis susceptibility loci to chromosomes 4 and 6 using the Ldlr knockout mouse model.

Atherosclerosis is a complex disease resulting from the interaction of multiple genes. We have used the Ldlr knockout mouse model in an interspecific genetic cross to map atherosclerosis susceptibility loci. A total of 174 (MOLF/Ei x B6.129S7-Ldlr(tm1Her)) x C57BL/6J-Ldlr(tm1Her) backcross mice, homozygous for the Ldlr null allele, were fed a Western-type diet for 3 months and then killed for quantification of aortic lesions. A genome scan was carried out by using DNA pools and microsatellite markers spaced at approximately 18-centimorgan intervals. Quantitative trait locus analysis of individual backcross mice confirmed linkages to chromosomes 4 (Athsq1, logarithm of odds = 6.2) and 6 (Athsq2, logarithm of odds = 6.7). Athsq1 affected lesions in females only whereas Athsq2 affected both sexes. Among females, the loci accounted for approximately 50% of the total variance of lesion area. The susceptible allele at Athsq1 was derived from the MOLF/Ei genome whereas the susceptible allele at Athsq2 was derived from C57BL/6J. Inheritance of susceptible alleles at both loci conferred a 2-fold difference in lesion area, suggesting an additive effect of Athsq1 and Athsq2. No associations were observed between the quantitative trait loci and levels of plasma total cholesterol, high density lipoprotein cholesterol, non-high density lipoprotein cholesterol, insulin, or body weight. We provide strong evidence for complex inheritance of atherosclerosis in mice with elevated plasma low density lipoprotein cholesterol and show a major influence of nonlipoprotein-related factors on disease susceptibility. Athsq1 and Athsq2 represent candidate susceptibility loci for human atherosclerosis, most likely residing on chromosomes 1p36--32 and 12p13--12, respectively.

Adrenocortical lipid depletion gene (ald) in AKR mice is associated with an acyl-CoA:cholesterol acyltransferase (ACAT) mutation.

ald, a recessive allele in AKR inbred mice, is responsible for complete adrenocortical lipid depletion in postpubertal males, which appears to be androgen dependent. Two recent observations (adrenocortical lipid depletion in acyl-CoA:cholesterol acyltransferase-deficient (Acact-/-) mice and the mapping of Acact to a region of chromosome 1 containing the ald locus) prompted us to ask whether adrenocortical lipid depletion in AKR mice results from an Acact mutation. Refined genetic mapping of Acact and ald was consistent with colocalization of these loci. Crossing Acact-/- with AKR (ald/ald) mice yielded postpubertal male offspring characterized by adrenocortical lipid depletion, indicating that these loci are not complementational and are therefore allelic. Immunoblotting of preputial gland homogenates demonstrated that AKR mice had an ACAT protein with a lower molecular mass than other mouse strains. Analysis of Acact cDNA from AKR mice revealed a deletion of the first coding exon and two missense mutations. Despite these coding sequence differences, the ACAT protein from the ald allele catalyzed cholesterol esterification activity at levels similar to that of wild-type protein. We speculate that the adrenocortical lipid depletion resulting from the ald mutation is caused by an altered susceptibility of the mutant protein to modifying factors, such as androgen production at puberty, in an as yet undetermined manner.

Genetic regulation of cholesterol homeostasis: chromosomal organization of candidate genes.

As part of an effort to dissect the genetic factors involved in cholesterol homeostasis in the mouse model, we report the mapping of 12 new candidate genes using linkage analysis. The genes include: cytoplasmic HMG-CoA synthase (Hmgcs 1, Chr 13), mitochondrial synthase (Hmgcs 2, Chr 3), a synthase-related sequence (Hmgcs 1-rs, Chr 12), mevalonate kinase (Mvk, Chr 5), farnesyl diphosphate synthase (Fdps, Chr 3), squalene synthase (Fdft 1, Chr 14), acyl-CoA:cholesterol acyltransferase (Acact, Chr 1), sterol regulatory element binding protein-1 (Srebf1, Chr 8) and -2 (Srebf2, Chr 15), apolipoprotein A-I regulatory protein (Tcfcoup2, Chr 7), low density receptor-related protein-related sequence (Lrp-rs, Chr 10), and Lrp-associated protein (Lrpap 1, Chr 5). In addition, the map positions for several lipoprotein receptor genes were refined. These genes include: low density lipoprotein receptor (Ldlr, Chr 9), very low density lipoprotein receptor (Vldlr, Chr 19), and glycoprotein 330 (Gp330, Chr 2). Some of these candidate genes are located within previously defined chromosomal regions (quantitative trait loci, QTLs) contributing to plasma lipoprotein levels, and Acact maps near a mouse mutation, ald, resulting in depletion of cholesteryl esters in the adrenals. The combined use of QTL and candidate gene mapping provides a powerful means of dissecting complex traits such as cholesterol homeostasis.

Involvement of the tyrosinase gene in the deposition of cardiac lipofuscin in mice. Association with aortic fatty streak development.

Lipofuscin pigment, a terminal oxidation product, accumulates within cells during the normal aging process and under certain pathological conditions. We have analyzed a genetic cross between two inbred mouse strains, BALB/cJ and a subline of C57BL/6J, which differ in lipofuscin deposition. A comparison of the segregation pattern of cardiac lipofuscin with the albino locus (c) on mouse chromosome 7 revealed complete concordance. Analysis of spontaneous mutants of the tyrosinase gene, encoded by the albino locus, confirmed that the tyrosinase gene itself controls lipofuscin formation. Genetic analysis of other strains indicated that one or more additional genes cab contribute to the inheritance of lipofuscin. We also present evidence for an association between cardiac lipofuscin deposition and aortic fatty streak development in the mouse.

Spectrophotometry of occult blood in feces.

This method for measuring fecal occult blood is based on the heme-catalyzed oxidation of tetramethylbenzidine by H2O2. An aliquot of heated stool homogenate is mixed with acetic acid to chemically separate heme from globin. The heme is extracted into ethyl acetate and reacted with the reagent and H2O2 to produce a green oxidation product. The reaction is followed kinetically for 30 to 60 s at 660 nm. A660 is linearly related to the amount of hemoglobin. The lower limit of detection is 1 to 2 mg of hemoglobin per gram (wet weight) of feces. Within-day precision (CV) of the analysis for hemoglobin added to stool specimens (4 to 30 mg/g) ranged from 2.3 to 7.6%, between-day CV from 2.1 to 8.1%. Analytical recovery of hemoglobin added to fecal specimens (4 to 30 mg/g) ranged from 86.7 to 106.2%. Of the substances known to interfere with conventional dye-oxidation tests for fecal occult blood, only myoglobin and ascorbic acid interfere with hemoglobin quantification by our procedure. The test is fast, inexpensive, and easy to perform, and involves equipment available in hospital laboratories.

Liquid-chromatographic determination of zomepirac in serum and plasma.

We describe a determination of zomepirac concentration in plasma and serum by reversed-phase "high-performance" liquid chromatography. The assay requires 1.0 mL of sample and involves diethyl ether extraction of zomepirac from an acidified sample, followed by concentration and injection into a liquid chromatograph. The column effluent is monitored at 330 nm. Retention times of zomepirac and the internal standard (tolmetin) are 3.8 and 2.7 min, respectively. The lower limit of detection for zomepirac in serum or plasma is 0.05 mg/L. Within-day precision (CV) of analysis in plasma or serum with zomepirac added (0.1-10.0 mg/L) ranged from 1.4 to 6.7%; between-day CV varied from 1.4 to 7.5%. Analytical recovery of zomepirac (1.0 mg/L) from serum and plasma was 77.6 (SD 3.5)% and 80.4 (SD 5.2)%, respectively. Numerous commonly coadministered drugs did not interfere. The elimination half-life of the drug was 1.8 h, and the peak plasma concentration ranged from 1.1 to 2.4 mg/L. Peak and trough concentrations measured throughout five days of therapy imply no accumulation.

Uptake of glycine from L-alanylglycine into renal brush border vesicles.

Isolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel from L-Ala. [3H]Gly and D-Ala.[3H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg2+. The microvilli vesicles transiently accumulated radiolabel from L-Ala. [3H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular greater than intravesicular) Na+ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all 3H uptake from L-Ala. [3H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate of L-Ala.[3H]Gly hydrolysis to free alanine and [3H[glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel from L-Ala.[3H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate that L-Ala.[3H]Gly is hydrolyzed at the external surface of the microvilli with the [3H]glycine released being transported into the vesicles by a Na+ gradient-dependent system identical to that employed for free glycine. Microvilli vesicle uptake of radiolabel from D-Ala.[3H]Gly exhibited no Na+ dependent "over-shoot" effect. D-Ala.[3H]Gly was completely resistant to microvilli-catalyzed hydrolysis. Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range of L-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli. Renal dipeptidase catalyzed hydrolysis of L-Ala.Gly but not D-Ala.Gly, as was the case with microvilli-catalyzed hydrolysis of the dipeptides. With its location in the renal brush border microvilli and its hydrolytic action against L-dipeptides, renal dipeptidase my act at the luminal surface of the proximal tubule cell to hydrolyze L-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na+ gradient-dependent processes.

Lipids of cultured hepatoma cells: VIII. Utilization of D-[1-14C] glucose for lipid biosynthesis.

Minimal deviation hepatoma 7288C cells (HTC) were incubated in serum-supplemented and serum-free Swim's 77 medium in the presence of D-[1-14C] glucose for 1, 2, 4, 8, 12 and 24 hr. Glucose oxidation to CO2, incorporation into total cell mass, and incorporation into cell and medium lipids were determined. The percentage distribution of total cell lipid radioactivity in individual neutral and polar lipid classes was followed as a function of time. Degradation studies of individual lipid classes were performed to ascertain the percentage of radioactivity in acyl and glycerol moieties. The percentage of D-[1-14C] glucose oxidized to 14CO2, incorporated into cell matter and cell lipids was elevated in cells incubated in serum-free medium as opposed to serum-supplemented medium. The percentage distribution of total cell lipid radioactivity into individual neutral lipid classes from both serum-free and serum-supplemented cultures was as follows: sterols greater than triglycerides greater than free fatty acids greater than sterol esters. The percentage distribution of total cell lipid radioactivity into individual polar lipid classes of serum-supplemented cultures was as follows: phosphatidylcholine greater than phosphatidylinositol greater than sphingomyelin greater than phosphatidylethanolamine greater than phosphatidylserine. The distribution of glucose radiolabel into individual polar lipid classes of serum-free HTC cells was different from their serum-supplemented counterparts: sphingomyelin greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylserine. Glycerol from glyceride classes contained a higher percentage of radioactivity than the acyl moieties, with this percentage significantly elevated in serum-free cultures. The data indicate that, although glucose is a substrate for HTC cell lipids, other precursors present in the culture system also contribute to the lipid constituency of this hepatoma cell line.