A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase.

We present a rapid, sensitive enzymatic assay for chloramphenicol acetyltransferase (CAT) that does not require chromatography, HPLC, or autoradiography. The assay is based on the use of an inexpensive substrate, tritiated acetate, instead of [14C]chloramphenicol. The method is adapted from one originally used by de Crombrugghe et al. (1973) and by Shaw (1975), but with simplifications appropriate for routine use. In our hands, the method is as sensitive as the customary thin-layer chromatography assay and is far more efficient for the performance of many assays, both in terms of labor and expense.

Carrier detection in hemophilia A: a cooperative international study. I. The carrier phenotype.

Eight laboratories in six countries cooperated to clarify several issues concerning the phenotypes of heterozygous carriers of hemophilia "A." Plasma levels of factor VIII (F.VIII:C, formerly VIII:C) and von Willebrand factor (VWF:Ag, formerly VIIIR:Ag) of carriers and normal women were determined by various "in-house" methods; a single lyophilized plasma standard was used for all assays. Analysis of the collated data from 336 carriers (296 obligatory carriers and 40 sporadic carriers) and 137 normal women showed that there was no difference in the F.VIII:C levels of "paternal" carriers (women who had obtained the abnormal gene from their fathers) and "maternal" carriers. Neither was there a difference in the VWF:Ag levels of normal women and either type of carrier. Age was found to have a significant effect on both F.VIII:C and VWF:Ag, values being higher at very young and very old ages, the minima occurring in the 25- to 30-year range. ABO blood type had a striking effect. Women of types A, B, and AB (designated non-O in the study), both normals and carriers, had significantly higher levels of both factors than did women of type O. Analysis by laboratories showed that differences in mean levels of both factors between laboratories were highly significant. It was concluded that age, ABO blood type, and laboratory variation should be taken into account in carrier detection.

Carrier detection in hemophilia A: a cooperative international study. II. The efficacy of a universal discriminant.

Factor VIII (F.VIII) and von Willebrand factor (VWF):Ag data collected by eight laboratories on a total of 336 obligatory carriers of hemophilia A and 137 normal women were used to answer several questions concerning the construction of linear discriminants for carrier detection. It was found: that a "universal" linear discriminant can be constructed which is suitable for use in all laboratories and is nearly as effective as laboratory-specific discriminants; that inclusion of age and ABO blood type data improved the efficacy of these discriminants; that substitution of alternative assays for F.VIII and VWF:Ag did not generally improve the efficacy of the discriminants over that obtained using the bioassay for F.VIII:C and Laurell's immunoassay for VWF:Ag; that linear discriminants were far more effective than discriminants based on the F.VIII:C/VWF:Ag ratio. A step-wise procedure is given which any laboratory may follow in using the universal discriminant for carrier detection.

Application of molecular genetics to prenatal diagnosis and carrier detection in the hemophilias: some limitations.

Prenatal diagnosis and carrier detection in the hemophilias have received much attention in recent years. The error rate in prenatal diagnosis by fetoscopy is less than 1%; fetoscopy is not possible, however, until the second trimester of pregnancy. Carrier detection based on bioassays of plasma has an irreducible error rate (approximately 5%?), because of the "lyonization" phenomenon in heterozygous women, and the final results are always probabilistic. New DNA methods promise to alleviate these difficulties. Prenatal diagnosis can be accomplished in the first trimester. "Lyonization" is bypassed in carrier detection, and the results may sometimes be essentially nonprobabilistic. But the DNA methods have certain limitations of their own which are not widely appreciated. Aside from cost and the necessity to adopt a new technology, there are inherent genetic problems: mothers must be heterozygous for both a disease gene and a marker gene, final results are probabilistic if the marker gene lies outside the disease gene, and multiple marker genes are often in linkage disequilibrium. We have concluded that a clinical unit planning to use the DNA methods must also maintain the conventional methods at a high level of performance.

A multivariate analysis of familial associations of lipoprotein levels in the Lipid Research Clinics Collaborative Family Study: I. Familial correlation and regression analyses.

In view of the complex, intraindividual relationships among different lipoprotein levels (LDL-C, HDL-C, and VLDL-C), multivariate methods aimed at assessing joint familial associations and their possible determinants were performed in the white, random sample component of the Collaborative Lipid Research Clinics Family Study data (1,336 families with 5,097 subjects). After appropriate transformations and covariate adjustments of the data, several kinds of correlation and regression analyses were performed, taking into consideration variable family size and possible age and clinic differences. The association patterns across clinics and age strata were found to be homogeneous for the vast majority of comparisons. The results of multivariate analyses (especially the significant association of each lipoprotein among biological relatives), the persistence of parental levels as the best predictors for the same lipoprotein levels among the offspring, and the essentially unchanged partial correlation estimates as compared to ordinary correlations suggest strong influence of factors specific to each lipoprotein in the familial associations. But the highly significant intraindividual correlations and the nonnegligible cross-correlations among relatives also suggest the additional presence of common underlying factors for the familial associations, especially between HDL-C and VLDL-C and to a lesser extent between LDL-C and VLDL-C. The issues stemming from these analyses and the directions for further analyses are discussed.

The Collaborative Lipid Research Clinics Family Study: biological and cultural determinants of familial resemblance for plasma lipids and lipoproteins.

This paper reports on the biological and cultural determinants of total, LDL, and HDL cholesterol, and triglyceride (TC, LDL-C, HDL-C, TG) levels using a general linear model on randomly selected family data collected during 1975-1978 at nine North American Lipid Research Clinics. Initially, the analyses were clinic-specific to assess the importance of genetic and cultural transmission, marital resemblance, and other determinants of these traits and then were made jointly to identify the nature and sources of any heterogeneity between clinics. There was evidence of significant genetic and cultural factors for all traits in most clinics. Clinic heterogeneity was also significant, but excluding one clinic reduced the heterogeneity considerably. The genetic (h2) and cultural (c2) heritabilities for the remaining eight clinics were homogeneous with pooled estimates of h2 of .556 +/- .028, .539 +/- .028, .485 +/- .029, and .358 +/- .028, and of c2 of .029 +/- .006, .033 +/- .006, .075 +/- .008, and .089 +/- .009 for TC, LDL-C, HDL-C, and TG, respectively. Among the traits, HDL-C exhibited the most difference among clinics, and both HDL-C and TG showed the largest cultural heritability. The relevance of these and similar studies in a broader understanding of the determinants of plasma lipids and lipoproteins is discussed.

The Collaborative Lipid Research Clinics Program Family Study. II. Response rates, representativeness of the sample, and stability of lipid and lipoprotein levels.

The Collaborative Family Study (1975-1978), the third phase of the Lipid Research Clinics Program Population Studies, covers 2405 probands and 15,693 of their relatives from nine North American communities. This sample was examined for participation differences across race, sex, locality, educational level, and reason for selection. The participation rates were somewhat lower for blacks, younger age groups, and subjects with lower educational levels. The probands' reason for selection into the study had little impact on the participation of probands or relatives. Moreover, based on information gathered at earlier examinations on eligible Family Study probands, the cornary risk factor profile appeared to be similar among participants and nonparticipants . The available longitudinal lipid data on probands indicated general consistency in lipid levels within subjects over short periods of time, in cholesterol even more so than in triglycerides. Among age strata, the younger subjects showed the least intrapersonal stability, especially for triglycerides.

The Collaborative Lipid Research Clinics Program Family Study. III. Transformations and covariate adjustments of lipid and lipoprotein levels.

Several methods of transformation and covariate adjustment have been applied to the Collaborative Lipid Research Clinics Program Family Study data to facilitate analysis of lipid levels of examinees of different sex and age groups. After several exploratory analyses, cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol were logarithmically transformed, and triglycerides were transformed by the power -0.25. Two types of covariate adjustment procedures, the regression and Z score methods, were used. A modified regression method was developed and was found to be preferable to both simple cubic regression and the Z score method on theoretical and empirical grounds. Refinements in this method to correct for change in variance with age, and for the effect of socioeconomic status, seasonality, and anthropometric measures were made. Methodological issues connected with the transformation and adjustment procedures are discussed.

The Collaborative Lipid Research Clinics Program Family Study. IV. Familial associations of plasma lipids and lipoproteins.

Familial associations of total cholesterol, low density lipoprotein cholesterol, triglycerides, and very low density lipoprotein cholesterol were examined in a population-based random sample of 858 white and 73 black probands and their 4,027 white and 245 black relatives from nine North American Lipid Research Clinics. Correlations among biologic relatives were highly significant for total cholesterol and low density lipoprotein cholesterol and to a lesser extent for triglycerides and very low density lipoprotein cholesterol in whites. Correlations for spouses, however, were not significant, suggesting a stronger influence of genes than shared environment in the determination of these traits. Homogeneity of familial correlations across age strata, clinics, and racial groups was examined. In general, correlations were homogeneous across age strata and clinics, and there was no asymmetry in parent-offspring correlations by the sex of the parent or offspring. Racial differences in correlations were not significant except in four of 32 comparisons, with blacks showing weaker correlations than whites in those instances.

The constancy of parent-offspring similarity of total cholesterol throughout childhood and early adult life. The Lipid Research Clinics Program Prevalence Study.

In a cross-sectional study of 11,409 white parent-offspring pairs in five North American populations we examined the effect of age of the offspring on parent-offspring total cholesterol correlations. In general there were no differences in correlations by age of the offspring for the four types (by gender) of parent-offspring pairs. This was true within each of the five populations and for the average of all populations combined. For offspring from less than 2-29 years of age, these average age-specific correlations ranged from 0.17 to 0.42. Despite the considerable physiologic and environmental changes which influence cholesterol levels from birth to early adulthood, the strength of parent-offspring similarity shows no consistent pattern of change.

Family aggregation of high density lipoprotein cholesterol. Collaborative lipid research clinics program family study.

High density lipoprotein cholesterol data on a population-based random sample of 858 white and 72 black probands and their 3935 white and 205 black relatives were collected from nine North American clinics using a common protocol and standardized methodology. Familial associations were examined within clinics for whites and pooled across clinics for blacks. The influence of covariates and varying family size on correlations was examined using several sets of transformed and adjusted values and a variety of weighting schemes. Parent-offspring and sibling correlations were significant in most cases, but spouse correlations were not, suggesting a stronger influence of shared genes than shared environment on high density lipoprotein cholesterol. Adjustment for covariates tended to weaken the correlations, but the effect of variable family size was imperceptible. Although pairs involving pediatric offspring or siblings tended to show higher correlation than their adult counterparts, the differences were not significant. All correlations except father-daughter and brother-brother were homogeneous across clinics in whites. There was no asymmetry in parent-child correlations by the sex of the offspring, but the pooled mother-child correlation was significantly higher than father-child values, suggesting a possible maternal influence on high density lipoprotein cholesterol. No heterogeneity in correlations in high density lipoprotein cholesterol was detected between blacks and whites except for mother-son pairs.