Comparison of serum and salivary cotinine measurements by a sensitive high-performance liquid chromatography-tandem mass spectrometry method as an indicator of exposure to tobacco smoke among smokers and nonsmokers.

Exposure to tobacco smoke, both from active smoking and from passive exposure to environmental tobacco smoke, can be monitored by measuring cotinine, a metabolite of nicotine, in a variety of biological sources including blood, urine, and saliva. Previously, a sensitive atmospheric-pressure ionization, tandem mass spectrometric (LC-API-MS-MS) method for cotinine measurements in serum was developed in support of a large, recurrent national epidemiologic investigation. The current study examined the application of this LC-API-MS-MS method to both serum and saliva cotinine measurements in a group of 200 healthy adults, including both smokers and nonsmokers. The primary objective of this study was to evaluate the relationship between serum and saliva cotinine concentrations to facilitate the linking of results from epidemiologic studies using salivary cotinine measurements to existing national data based on serum cotinine analyses. The results indicate that a simple, linear relationship can be developed to describe serum and saliva cotinine concentrations in an individual, and the expression describing this relationship can be used to estimate with reasonable accuracy (approximately +/- 10%) the serum cotinine concentration in an individual given his or her salivary cotinine result. It was further confirmed that saliva cotinine samples are generally quite stable during storage after collection, even at ambient temperatures, and this sample matrix appears to be well-suited to the requirements of many epidemiologic investigations.

Determination of "free" glycerol in human serum reference materials by isotope-dilution gas chromatography-mass spectrometry.

Serum free glycerol analyses are an important part of the preparation and evaluation of human serum reference materials used for the quality assurance of triglyceride assays. However, enzymatic kits for free glycerol analysis obtained from different vendors have, on occasion, provided different results for a given sample. In an effort to establish the "target" glycerol content of selected reference materials, we have established a method for the analysis of serum free glycerol by using isotope-dilution gas chromatography-mass spectrometry, incorporating [1,3-13C2]glycerol as the internal standard. The use of a simplified serum extraction and clean-up procedure resulted in (uncorrected) recoveries of glycerol averaging about 90% before derivatization, and in estimated concentrations for spiked serum pools that corresponded closely to the expected values. A comparison of enzymatic and gas chromatographic-mass spectrometric results for several reference serum pools suggest that the latter method is of value in evaluating and validating routine enzymatic methods for free glycerol analysis.

A case of hemoglobin Indianapolis [beta 112(G14) Cys----Arg] in an individual from Cordoba, Spain.

Hemoglobin Indianapolis was first described by Adams et al (1,2) as a very unstable variant with a phenotype similar to severe beta-thalassemia. We have also characterized this variant, but there are several differences in the clinical expression of the variant described in our report and those described in the original case. We found Hb Indianapolis to be unstable, but not to the extent that it could not be detected by routine testing. The four family members heterozygous for the variant were not anemic, showed normal hematologic values, and did not exhibit any severe clinical disadvantages, although there was slight reticulocytosis. The variant could not be resolved from Hb A on cellulose acetate (pH 8.4), but isoelectric focusing showed a double band in the region of Hb A that is probably the variant and Hb A. However, the variant chain was clearly evident by globin chain analyses in acid and alkaline buffers. The condition of additional blood samples did not allow us to determine the oxygen dissociation properties of the variant or the rates of globin chain synthesis.

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis.

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.

Hemoglobin Shelby [beta 131(H9) Gln----Lys] a correction to the structure of hemoglobin Deaconess and hemoglobin Leslie.

Hemoglobin Shelby, detected in two unrelated black families, has an electrophoretic mobility like Hb F on cellulose acetate (pH 8.4) and a mobility between Hbs S and C on citrate agar (pH 6.2). Globin chain analysis in acid and alkaline buffers revealed an abnormal chain migrating between beta A and beta S. Tests for unstable hemoglobins were positive. Hematologic data on both families indicated carriers have mild anemia. The variant showed a slightly lower affinity for oxygen with normal cooperativity and Bohr effect, and its reactions with 2,3-diphosphoglycerate and inositol hexaphosphate were similar to those of Hb A. Sequence analysis indicated the substitution of lysine for glutamine at position 131 in the beta-chain. In a previous report (1) we described a variant, Hb Deaconess, in which this residue was deleted. On reexamination of the data, we find that Hb Deaconess is identical to Hb Shelby.

Hemoglobin Evanston: alpha 14(A12) Trp leads to Arg. A variant hemoglobin associated with alpha-thalassemia-2.

Hb Evanston (alpha 14 Trp leads to Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the alpha-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the alpha-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with alpha-thalassemia-2 produced anemia in this black family.

Hemoglobin Cheverly: an unstable hemoglobin associated with chronic mild anemia.

The evaluation of a family with chronic mild anemia led to the identification of a new unstable hemoglobin (Hemoglobin Cheverly). Modest anemia and reticulocytosis, normal to slightly increased mean corpuscular volume (MCV), and normal mean corpuscular hemoglobin concentration (MCHC) were present in the affected family members. Electrophoresis of blood samples on cellulose acetate and on citrate agar revealed normal patterns. Globin chain analysis and isoelectric focusing data were also normal. After incubation for 3 h at 41 degrees C, Heinz bodies were detected in 95-100% of erythrocytes from affected individuals. Positive heat and isopropanol tests confirmed the initial observation of the Heinz body preparation and indicated that an unstable hemoglobin was present. Structural analysis showed an amino acid substitution of Phe-Ser at position 45 (CD4) in the beta chain. Hemoglobin Cheverly has a reduced affinity for oxygen and a reduced Bohr effect, properties that can be rationalized on the basis of the x-ray crystallographic structure of normal hemoglobin. Despite structural and functional similarities between Hb Cheverly and Hb Hammersmith, beta 42 (CD1) Phe-Ser, the clinical manifestations of Hb Cheverly are mild in contrast to the severe disease observed with Hb Hammersmith. Reasons for the apparently silent clinical expression of Hb Cheverly are not known. We discuss the implications of unstable hemoglobins in the evaluation of chronic anemia in pediatric patients.

Hemoglobin Queens: alpha 34 (B15) Leu-Arg structural and functional properties and its association with Hb E.

Hemoglobin Queens: alpha 34 (B15) Leu-Arg was found in association with Hb E in a Vietnamese boy. The observed microcytosis and hypochromia were due to Hb E, which also occurred in other members of this family. The functional properties of Hb Queens in the absence and presence of allosteric effectors were normal. The abnormal hemoglobins appear to be unrelated to the hyperbilirubinemia that was observed at birth.