Dapsone at onset of diabetes lowers glycated hemoglobin and delays death in NOD mice.

Dapsone (4,4'-diaminodiphenyl sulfone) is a compound that has a large clinical experience due to its antimicrobial effects against mycobacterium leprae, the causative agent of leprosy. It is increasingly used in a number of clinical situations where inflammation may play an ancillary role. An inhibitory effect of the drug or lack thereof in the cumulative incidence or propagation of diabetes mellitus in the NOD mouse has mechanistic as well as therapeutic implications. We previously showed that dapsone administered continuously as a percentage of food to NOD mice inhibits the cumulative incidence of diabetes in a dose dependent fashion. In the present experiment, female NOD litter mates were randomized to receive dapsone (0.001% w/w as a percentage of food) at onset of diabetes. There were no differences in weight, blood glucose, or glycated hemoglobin at 10 weeks of age among the animals that were ultimately to receive dapsone (n = 10), mouse chow alone (n = 9), or those who did not develop diabetes (n = 3). The mean time to onset of diabetes, mean blood glucose at onset, and mean glycated hemoglobin at onset did not differ between animals who did or did not receive dapsone. Animals receiving dapsone had significantly (p < or = 0.03) lower glycated hemoglobin at weeks 2, 3, and 4 following the onset of diabetes and lived significantly longer following diagnosis of diabetes (7 vs. 4 weeks, p < 0.05). In conclusion, dapsone modulates the progression of autoimmune diabetes in the NOD mouse even when administered after the initiation of hyperglycemia.

Validated fluorimetric HPLC analysis of acetaldehyde in hemoglobin fractions separated by cation exchange chromatography: three new peaks associated with acetaldehyde.

Stable hemoglobin-acetaldehyde adducts present in hemoglobin fractions separated by polyaspartic acid cation exchange chromatography were quantified by fluorimetric HPLC. The fluorescent species eluted from the HPLC was confirmed by mass spectrometry to be consistent with the expected product from reaction of acetaldehyde, 1,3-cyclohexanedione (CHD), and ammonium ion. Hemolysate (2.2 mM hemoglobin) was incubated in equivalent volumes of either phosphate-buffered saline or 5 mM acetaldehyde at 37 degrees C for 30 min and washed three times with H2O to remove free acetaldehyde and labile adducts before the injection of 14.7 mg hemoglobin onto the cation exchange column. Fluorimetric HPLC analysis of hemolysate samples either with or without in vitro reaction with acetaldehyde revealed that most acetaldehyde resides in the hemoglobin A0 fraction. The reaction with acetaldehyde in vitro resulted in a significant increase in fast-eluting minor hemoglobin species on cation exchange chromatography concomitant with increased acetaldehyde in the HbA1a+b, HbA1c, and HbA1-AcH fractions. We report three new cation exchange chromatographic peaks after reaction with acetaldehyde: HbA1-AcH-3, HbA1c-1, and HbA0-1. Each new peak was found to associate with a significant quantity of CHD-reactive acetaldehyde. These experiments provide additional evidence that stable adducts form between acetaldehyde and hemoglobin and that these adducts occur in multiple hemoglobin species separated by cation exchange chromatography. Further characterization and structural assignment of these species are warranted in view of their potential utility as markers for ethanol intake.

Peak running velocity is highly related to distance running performance.

This study examined the relationship between the peak running velocity (PRV) obtained during a horizontal, incremental treadmill test and distance running performance in a group of highly-trained male (N = 14) and female (N = 9) distance runners. Performance (5 km run time) was assessed with a self-paced time trial under laboratory conditions in an attempt to minimize extraneous variables which could affect performance (i.e. environmental conditions, terrain, etc.); relationships with recent best 5 km race time were also determined. PRV was highly related to 5 km performance whether determined from the time trial (r2 = 0.94, p < 0.001) or recent race (r2 = 0.89, p < 0.001). A new finding was that PRV was similarly related to performance in both the male (r2 = 0.83, p < 0.001) and female (r2 = 0.80, p < 0.001) athletes. Peak running velocity is thus highly predictive of distance running performance in highly-trained endurance runners. This finding has important practical implications, as PRV can be measured without extensive metabolic equipment and/or invasive techniques.

Studies of the oxidation of ethanol to acetaldehyde by oxyhemoglobin using fluorigenic high-performance liquid chromatography.

We noted a rise in acetaldehyde levels in clinical samples of venous whole blood containing ethanol that did not occur in samples from teetotalers. Experiments were performed to define the mechanism involved in acetaldehyde production. The addition of 0.10% ethanol to whole blood produced an immediate increase in acetaldehyde due to acetaldehyde in the stock solution followed by a subsequent increase that became statistically significant by 48 hr. Separation of blood into components documented that the increase in acetaldehyde was associated with the red cell but not plasma fraction. Incubation of isolated hemoglobin with ethanol produced a rise in acetaldehyde levels. Incubation of oxygenated whole blood with ethanol produced a linear increase in acetaldehyde, whereas nitrogen-exposed blood produced no increase. The rise of acetaldehyde in the presence of ethanol was dependent on the concentration of oxygenated hemoglobin A0. Addition of inhibitors of catalase, alcohol dehydrogenase, and glycolytic enzymes (aminotriazole, azide, pyrazole, sodium fluoride, sodium citrate, and iodoacetate) did not inhibit the rise of acetaldehyde, but addition of the hemoglobin ligand cyanide abolished the rise in acetaldehyde. Kinetic analysis with oxygenated whole blood plus inhibitors revealed a Km of 2.5 mM and Vmax of 1.42 microM/min. We conclude that oxyhemoglobin contributes to the metabolism of ethanol to acetaldehyde. These findings may explain in part the high levels of acetaldehyde found in red cells compared with plasma. The results also have implications for the optimum storage of blood samples for acetaldehyde analysis.

The effects of taper on performance in distance runners.

The purpose of this study was to determine if a 7-d systematic reduction in training volume or "taper" could improve distance running performance. Three groups of eight runners were examined: 1) run taper, 2) cycle taper, and 3) control. Training in the run taper group consisted of high-intensity intervals and an 85% reduction in training volume. The cycle taper group performed an equivalent amount of interval training as the run taper group, but each member exercised on a cycle ergometer. Control subjects continued normal training. A self-paced 5-km time trial served as the index of performance. The run taper group decreased 5-km time by 3% (1036.2 +/- 30.6 to 1006.8 +/- 28.2 s, P < 0.005). A significant decrease (P < 0.01) in submaximal oxygen consumption (6%) and calculated caloric expenditure (7%) at a running speed eliciting 80% of VO2max was also evident in the run taper group. Five-km performance and running economy were not altered in the cycle taper or control groups. These findings indicate that 7 d of tapered running improved distance running performance and running economy. A taper regimen of equivalent duration cycle training maintained performance in distance runners.

Sex differences in glycated hemoglobin in diabetic and non-diabetic C57BL/6 mice.

Pathophysiological implications of gender may be important in a number of disease states. We therefore decided to study the influence of gender on glycation in mice. Plasma glucose and glycated hemoglobin levels were determined by ion exchange (HbA1c) and/or affinity chromatography (GHb) in C57BL/6 ob/ob mice during the onset and subsequent decline of hyperglycemia. In preweanling ob/ob mice, glucose and glycated hemoglobin concentrations were equal to those of lean sex-matched controls. Shortly after weaning, plasma glucose in ob/ob mice increased to reach a maximum between 2 and 3 months of age, then declined over the next several weeks to levels within the range of lean mice. HbA1c values were closely associated with the glycemic changes. Male mice of both phenotypes consistently had higher values of glycated hemoglobin at a given glucose value than did females. Disappearance rates of chromium-labeled erythrocytes were slightly higher in lean female mice than in other subgroups but after correcting for phenotype/sex differences in blood volume, no phenotype or gender differences in RBC lifespan were observed. We conclude that there are gender differences in glycation of hemoglobin in mice and that factors other than RBC turnover are associated with the gender effects in both obese and lean mice.

Breath acetaldehyde following ethanol consumption.

Five pairs of volunteers were studied to determine the effect of drinking ethanol on breath acetaldehyde levels. On a given study day, samples of breath were obtained for measurement of acetaldehyde and ethanol from both participants at t = -1 h, t = -0.5 h, and at t = 0 to obtain baseline values. The drinkers were then given ethanol (0.3 g/kg body weight), and the controls given an equal volume of tap water. Breath samples were then taken at 0.5, 1, 1.5, 2 h, and hourly until t = 6 h. The last sample taken was at t = 23.5 h. Acetaldehyde levels in breath were quantified with a fluorigenic high-performance liquid chromatographic assay. Blood ethanol was approximated using a breath analyzer. Acetaldehyde in breath rose 50-fold at the 0.5-h, time point and returned to levels not significantly different from baseline values by 3-4 h. The mean peak blood ethanol values reached 0.055%. The t 1/2 elimination for ethanol was 1.6 h, and that for acetaldehyde was 2.25 h. Elimination of both acetaldehyde and ethanol in breath were initially 0 order. A significant correlation (r = 0.74) was found between baseline breath acetaldehyde levels and peak acetaldehyde levels. We conclude that acetaldehyde resulting from ethanol intake rapidly partitions into breath. The correlation of baseline breath acetaldehyde values with peak values found after an ethanol challenge indicate that measurement of breath acetaldehyde may be useful in the identification of individual differences in ethanol metabolism.

Studies of urine-associated acetaldehyde as a marker for alcohol intake in mice.

A study was undertaken in 16 male C57BL mice to evaluate the effect of ethanol intake via the drinking water (10% v/v) on urinary-associated acetaldehyde. Eight received ethanol and 8 served as controls. Urinary-associated acetaldehyde (UAA) was measured using a fluorigenic high performance chromatographic assay. Ethanol consumption did not impair growth over the two weeks of the experiment. Following administration of ethanol, UAA increased and remained significantly elevated over levels seen in controls until ethanol administration ceased (11.3 +/- 3.6 SEM microM for ethanol-consuming mice vs. 0.69 +/- 0.33 microM for controls). Ethanol in the urine was found to interfere with the assay for acetaldehyde. However, following cessation of ethanol, acetaldehyde in urine was found to be significantly elevated in urine at 24 hours, after ethanol levels were no longer detectable. In conclusion, measurement of urinary-associated acetaldehyde discriminates ethanol-consuming from nonconsuming mice during ethanol ingestion as well as 24 hours following cessation of ethanol when ethanol levels are no longer detectable in urine. Thus measurement of urinary acetaldehyde may be a useful marker for monitoring ethanol intake.

Hemoglobin associated acetaldehyde correlates with the Self-Administered Alcoholism Screening Test but not glycated hemoglobin in type II diabetes mellitus.

Twenty seven subjects with Type II diabetes mellitus underwent analysis of the Self-Administered Alcoholism Screening Test (SAAST) and hemoglobin associated acetaldehyde levels (HbAA). The SAAST scores and HbAA levels correlated with one another (r = .48, p = 0.009). No correlation between glycated hemoglobin levels (GHb) and HbAA levels or SAAST was found. Glucose incubation of whole blood led to an increase in GHb but no change in HbAA. We conclude that HbAA and SAAST correlate with each other when measured in patients with diabetes. Therefore each test appears clinically useful in quantifying alcohol consumption in individuals with Type II diabetes mellitus.

Studies of whole blood associated acetaldehyde as a marker for alcohol intake: effect of gender in mice.

A study was undertaken in C57BL mice to evaluate the effect of gender on whole blood associated acetaldehyde following exposure to ethanol in the drinking water (10% v/v). Whole blood associated acetaldehyde (WBAA) was measured from capillary blood samples using a fluorigenic high performance chromatographic assay on days 0, 7, 15 and 27. Ethanol consumption did not impair growth of either male or female mice when compared to controls. Following administration of ethanol, WBAA increased in both male and female mice but marked gender differences were apparent. Female mice consumed more fluid relative to body weight than males (155 +/- 27 S.D. vs. 124 +/- 19 ml/kg/day, p less than 0.001), but had lower mean WBAA levels during the four weeks of ethanol administration (137 +/- 37 vs. 318 +/- 66 nmol/g hemoglobin, p less than 0.001). WBAA levels in male mice were stable over the course of the experiment. Female mice were found to have peak WBAA levels on day seven after which time levels decreased significantly. These experiments emphasize gender differences in ethanol metabolism as well as the need to establish norms based on gender for assays of ethanol consumption which use acetaldehyde adducts with blood proteins.

A comparative blinded study in miniature swine of whole blood-, hemoglobin-, platelet-, plasma-, and lymphocyte-associated acetaldehyde as markers for ethanol intake.

Blood samples were obtained from miniature swine maintained on 0, 2, or 6 g/kg/24 hr ethanol for 8 months (N = 6 in each group). Samples from drinking pigs were taken after 8 hr of ethanol abstinence and all were coded and sent for "blinded" analysis. A fluorigenic high performance liquid chromatographic assay was used to quantify whole blood-associated acetaldehyde, hemoglobin-associated acetaldehyde, plasma-associated acetaldehyde, platelet-associated acetaldehyde, and lymphocyte-associated acetaldehyde. Detectable levels of acetaldehyde were found in each sample in both drinking and nondrinking pigs. Analysis of whole blood-associated acetaldehyde was most discriminatory in distinguishing nondrinking from drinking pigs (mean 21.4 +/- 1.0 microM for nondrinkers vs. 24.6 +/- 1.5 SD for the group consuming 2 g/kg ethanol, p = 0.001). Measurements of hemoglobin-associated acetaldehyde normalized to protein concentration (250 +/- 47 nmoles/g vs. 203 +/- 33 SD, p less than 0.05 drinking vs. nondrinking pigs) and platelet-associated acetaldehyde (0.46 0.34 vs. 0.15 +/- 0.16 nmoles/3 x 10(8) platelets, p = 0.05 drinking vs. nondrinking pigs) were also useful in discriminating drinking from nondrinking animals. Analysis of plasma-associated acetaldehyde and lymphocyte-associated acetaldehyde were not useful as markers of ethanol consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cigarette smoking on breath and whole blood-associated acetaldehyde.

Six pairs (1 habitual smoker and 1 nonsmoking control) of volunteers were studied to determine the effect of smoking tobacco on breath and whole blood acetaldehyde levels. On a given study day, samples of blood and breath were obtained from both participants at -0.25, 0, 0.25, 0.50, 0.75, 1.5, 2.5, and 3.5 hour time points. The smoking volunteer was told to smoke 1-3 cigarettes between the 0 and 0.25 hour time points. Acetaldehyde levels in breath and whole blood were quantified with a fluorigenic high performance liquid chromatographic assay. Acetaldehyde in breath rose six-fold in smokers at the 0.25 hour time point and returned to levels not significantly different from baseline values found in smokers or nonsmokers by 0.50 hr. Whole blood-associated acetaldehyde measurements remained unchanged in smokers throughout the experiment and were not different from nonsmokers. In conclusion, while smoking produces appreciable levels of acetaldehyde in expired air, the partitioning of acetaldehyde associated with smoking to blood or blood proteins appears to be below the level of detection of the assay employed (picomolar). Smoking of tobacco products may not interfere with assays designed to quantify ethanol intake by measuring acetaldehyde adducts with blood proteins.

Correlation of self-administered alcoholism screening test with hemoglobin-associated acetaldehyde.

Thirty subjects underwent analysis of the Self-Administered Alcoholism Screening Test (SAAST) and hemoglobin-associated acetaldehyde levels (HbAA). Eleven alcoholic individuals reporting for treatment, 10 self-defined "social drinkers" and 10 self-defined "teetotallers" who had consumed less than 6 drinks of ethanol per year ever in their lifetime and had not knowingly had ethanol for 6 months were included in the study. The SAAST scores and HbAA levels correlated with one another (r = .55, p = 0.002). For alcoholic individuals, the mean HbAA level was 180 +/- 64 nm/g Hb and the mean SAAST score was 17.3 +/- 6.3. Both analyses could distinguish the alcoholic from the teetotaller group (p = 0.03 for HbAA and p less than 0.001 for the SAAST) and from the group of social drinkers (p = 0.04 for HbAA and p less than 0.001 for the SAAST). The difference in HbAA levels between the social drinkers and teetotallers was not significant (132 +/- 25 vs. 129 +/- 15 nm/g Hb). After modification of the SAAST analysis for teetotallers, SAAST scores were significantly different between social drinkers and teetotallers (3.5 +/- 2.2 vs. 1.1 +/- 1.2, p = 0.009). We conclude that HbAA and SAAST correlate with each other may be clinically useful in distinguishing alcoholic from nonalcoholic individuals. While the SAAST appears to be the more sensitive test, it requires modification in the case of teetotallers.

Studies of whole blood associated acetaldehyde as a marker for alcohol intake in mice.

Thirty C57BI mice were randomized into two groups. Group 1 served as controls while Group 2 was given 10% V/V ethanol with the drinking water. Whole blood- associated acetaldehyde (WBAA) was measured on capillary blood samples using a fluorigenic high performance chromatographic assay. WBAA peaked at Day 2. A stable mean plateau of 263 +/- 71 SD with a range of 160-400 nmoles/g hemoglobin WBAA was found in the group consuming ethanol compared with 122 +/- 17 SD and a range of 88-150 nmoles/g hemoglobin for controls (p less than 0.001). When ethanol was discontinued, levels of WBAA declined and became similar to those of controls by 9 days following cessation of ethanol. The quantitative difference between ethanol-consuming and control animals and also the rapid rise of whole blood-associated acetaldehyde and the relatively slow decline following cessation of ethanol intake indicate that such a test might be a useful monitor of drinking behavior.

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse.

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.

Serological and biochemical identification of a plasma membrane antigen specific to Leydig cells.

Purified mouse Leydig cells have been prepared from interstitial cell suspensions using a Percoll gradient procedure. The isolated cells are 88-95% pure as determined by light microscopy. Staining for 3-beta-hydroxysteroid dehydrogenase indicates that over 85% of the Leydig cells recognized by differential interference microscopy are also positive for this enzyme. After separation, the Leydig cells are viable by dye exclusion assays and exhibit normal in situ morphology when examined at the ultrastructural level. Leydig cell suspensions have been used to raise a polyclonal antiserum in rabbits. This antiserum, prior to absorption, reacts in indirect immunofluorescent studies with the surfaces of isolated Leydig cells, with testicular germ cells, with spermatozoa and with somatic cells such as splenocytes. Following absorption with lymphocytes and spermatogenic cells, the antiserum binds only to Leydig cell plasma membranes. Quantitative measurements with 125I-protein A confirm the specific labeling of Leydig cells by the absorbed antiserum. Biochemical identification of the Leydig cell plasma membrane antigen has been accomplished by immunoblotting polyacrylamide gel nitrocellulose transfers. A single major band of Mr approximately 40,000 is detected on one-dimensional transfers; two weakly reactive spots of Mr 43,000 and 45,000 are detected using two-dimensional immunoblots. Blotting experiments conducted using concanavalin A have identified the major Leydig cell constituents reactive with this lectin. The Leydig cell plasma membrane antigen(s) does not bind concanavalin A.

Identification of spermatogenic cell plasma membrane glycoproteins by two-dimensional electrophoresis and lectin blotting.

Plasma membrane glycoproteins present in purified mouse spermatogenic cells have been identified by two-dimensional polyacrylamide gel electrophoresis and lectin blotting techniques. Four membrane glycoproteins labelled with Bandeiraea simplicifolia lectin (I) have been detected, ranging in Mr from 55 000 to 76 000 and in pI from 6.0 to 6.3. Only one of these proteins, p76/6.3, is synthesized by short-term in vitro cultures of spermatogenic cells, as determined by the incorporation of [35S]methionine. Approximately 20 surface glycoproteins labelled with concanavalin A have been identified, ranging in Mr from 50 000 to 151 000 and in pI from 5.7 to 7.0. None of the components detected with B. simplicifolia lectin (I) are labelled significantly with concanavalin A. A major concanavalin A-binding protein in the membranes of both pachytene spermatocytes and round spermatids is p151/6.0. This glycoprotein has been previously shown to be exposed on the outer surface of spermatogenic cell membranes and may represent a mediator of germ cell-Sertoli cell interactions. Furthermore, two constituents identified in the present study represent stage-specific markers. Component p73/5.7 is detected with concanavalin A only in the membranes of pachytene spermatocytes. Conversely, p84/6.3 is found only in round spermatid membranes. These results, then have: (a) provided a map of membrane glycoproteins in developing mouse male germ cells; (b) identified p151/6.0 as a membrane constituent of possible functional significance; and (c) identified the first reported glycoprotein surface differentiation markers for mouse spermatogenesis.