Transmenopausal changes in the trabecular bone structure.

INTRODUCTION: Post-menopausal osteoporosis is a disorder of excess skeletal fragility, due partly to changes in bone microstructure. Menopause is known to result in bone loss and reduction in bone mechanical strength. However, the mechanism and nature of microstructural changes at menopause need more detailed description and analyses. The overall hypothesis for this analysis is that the variables describing trabecular bone micro-architecture will be affected by changes in the hormonal status of women just prior to, and early after, last menses, and that volumetric bone density, and trabecular structure will decline significantly. The study was designed to capture true longitudinal transmenopausal changes in three-dimensional (3-D) trabecular bone architecture. Currently, minimal data exist regarding these features. MATERIALS AND METHODS: Transilial biopsies specimens were obtained from healthy pre-menopausal women (age >46), and repeated at 12 months after the last menstrual period. Bone architecture was quantified in 38 paired specimens using micro-computed tomography (micro-CT-40, Scanco) techniques. Bone biopsies were embedded for histomorphomteric analyses and parts of the analyses have been published elsewhere. Embedded bone biopsies were scanned at 30-mum resolution such that the region of interest was similar to that in the two-dimensional (2-D) histomorphometric analyses. Paired t-tests were used to compare the pre- and post-menopausal bone structural data from each technique. RESULTS: There was good correlation between standard histomorphometric (2-D) and micro-CT (3-D) measurements. Most of the variables characterizing bone structure in post-menopausal women (from micro-CT) significantly decreased (BV/TV, trabecular number, apparent and tissue density). In addition, both trabecular spacing (Tb.S) and the structure model index (SMI) increased in the post-menopausal women suggesting transformation of trabecular bone from plate- to rod-like structure. The 3-D trabecular connectivity density (Conn.D) was negatively correlated with activation frequency (Ac.f). CONCLUSIONS: These data suggest that 3-D micro-CT measurements (longitudinal) are comparable to those of standard histomorphometry, and that most of the bone structural measurements are sensitive to changes in women's hormonal status across menopause.

Factors influencing skeletal maturation at diagnosis of paediatric Cushing's disease.

BACKGROUND/AIMS: Growth retardation is a recognised complication of paediatric Cushing's disease (CD), but there are few published data on skeletal maturation at diagnosis. We assessed factors contributing to skeletal maturation in patients with paediatric CD. PATIENTS/METHODS: 17 patients, 12 males, 5 females (median age 12.1 years, range 5.8-17.4) were studied. The bone age (BA) of each child was determined by a single observer using the TW3 RUS method. BA delay, i.e. the difference between chronological age (CA) and BA, was compared with clinical and biochemical variables. RESULTS: BA delay was present in 15/17 patients (mean delay 2.0 years, range -0.5 to 4.1 years) and correlated negatively with height SDS (r = -0.70, p < 0.01) and positively with duration of symptoms (r = 0.48, p = 0.05) and CA (r = 0.48, p = 0.05). No relationships were found with midnight cortisol, ACTH, DHEA-S or cortisol suppression during the low-dose dexamethasone suppression test. CONCLUSIONS: BA in most children with CD was delayed and related to length of symptoms and height SDS at diagnosis. Early diagnosis will reduce delay in skeletal maturation and thus contribute to optimal catch-up growth.

Linear growth and body mass index in pediatric patients with Cushing's disease or simple obesity.

BACKGROUND: Increasing prevalence of childhood obesity has resulted in an accelerating rate of referrals of overweight patients to pediatric clinics for exclusion of endocrine or metabolic etiologies. The exclusion of Cushing's disease (CD) requires complex and potentially invasive investigations. OBJECTIVE: To evaluate the sensitivity of accurate measurements of height, weight and body mass index (BMI) in discriminating between simple obesity and CD. METHODS AND PATIENTS: Height, weight and BMI were measured at diagnosis in 25 patients with CD; 14 males, 11 females, mean age 12.9 yr (6.4-17.8) and 41 patients with simple obesity (SO), defined as BMI >2.0 SD; 20 males, 21 females, mean age 9.4 yr (3.5-15.6). RESULTS: Mean (+/-SE) BMI SDS in the CD patients was 2.41+/-0.5 and in the SO patients 3.71+/-1.3. Height SDS in the CD patients was -1.88+/-0.24 and in the SO patients 1.18+/-0.19 (p<0.05). The mean (+/-SE) BMI SDS to height SDS ratio was significantly decreased in the CD compared with the SO patients; -1.81+/-0.54 vs +0.90+/-1.17 (p<0.0001). CONCLUSIONS: Simple, accurate measurement of height and BMI SDS values provides a quick, and sensitive diagnostic discriminator in pediatric patients with CD or SO, thus potentially avoiding complex investigations.

Benefits of screening in von Hippel-Lindau disease--comparison of morbidity associated with initial tumours in affected parents and children.

Von Hippel-Lindau (VHL) is a rare autosomal dominant syndrome characterised by the association of retinal and CNS haemangioblastomas, phaeochromocytoma and renal cell carcinoma. If a child of an affected parent has inherited a VHL mutation or the parent's mutation cannot be identified, then clinical screening is recommended. We report the clinical features in three parent-offspring pairs where the parents have presented clinically with renal cell carcinoma, phaeochromocytoma, cerebellar haemangioblastoma and retinal haemangioma, and the children have undergone pre-symptomatic screening. During the first screening a 13-year-old boy was diagnosed with bilateral phaeochromocytoma and later developed an endolymphatic sac tumour at 19 years. A right phaeochromocytoma was found in a 12-year-old girl who was screened from the age of 4 years and in a 13-year-old boy screened from 5 years of age. All children were asymptomatic at the time of diagnosis. These families demonstrate that clinical screening of children at risk of VHL can detect tumours before the first symptoms arise with a consequent reduction in morbidity. These observations strongly support the recommendation to undertake screening of the children of VHL patients.

Bone mineral density at diagnosis and following successful treatment of pediatric Cushing's disease.

Bone mineral density (BMD) is frequently reduced in children and adolescents with Cushing's disease (CD), but there is little follow-up data after cure. BMD was determined by dual energy X-ray absorptiometry (DEXA) in two groups of patients with CD. Group 1 comprised 8 patients, 5 males and 3 females, aged 12.4 yr (8.2-16.8), assessed at diagnosis. Group 2 comprised 11 subjects, 6 males and 5 females, diagnosed at age 13.3 yr (6.4-17.4), cured by transsphenoidal surgery (TSS) (no.=7) or TSS + pituitary irradiation (no.=4). They had measurement of BMD, at mean age of 18.3 yr (11.1-28.5), i.e. 4.5 yr (0.8-11.4) after cure. Four patients, mean age 20.2 yr (17.6-22.4), had repeated DEXA'scans, 1-4 times, for up to 5.8 yr. After cure, GH deficiency was present in 9 patients and treated with hGH in 8. In Group 1, patients' L2-L4 volumetric (v)BMD Z-score was variable with a mean of -1.04 (-3.21-0.11). L2-L4 vBMD Z-score values correlated negatively with midnight cortisol (p < 0.05). In Group 2, mean L2-L4 vBMD was -0.38 (-1.0-0.13); and in 7/11, mean femoral neck (FN) areal (a)BMD Z-score was 0.14 (-1.62-2.46). FN aBMD Z-score was higher than L2-L4 aBMD Z-score (p < 0.05). In patients with repeated scans, mean change in L2-L4 vBMD Z-score was 0.20 (-0.15-0.45), and mean change in FN aBMD Z-score 0.03 (-0.53-0.38). These findings show variability of BMD at diagnosis and near normal BMD after cure of pediatric CD, suggesting that with appropriate replacement of pituitary hormone deficiency normal peak bone mass is achievable.

A genome-wide linkage scan for bone mineral density in an extended sample: evidence for linkage on 11q23 and Xq27.

BACKGROUND: Osteoporosis is a major public health problem, mainly quantified by low bone mineral density (BMD). The majority of BMD variation is determined by genetic effects. A pilot whole genome linkage scan (WGS) was previously reported in 53 white pedigrees with 630 subjects. Several genomic regions were suggested to be linked to BMD variation. OBJECTIVE: To substantiate these previous findings and detect new genomic regions. METHODS: A WGS was conducted on an extended sample where the size was almost tripled (1816 subjects from 79 pedigrees). All the subjects were genotyped with 451 microsatellite markers spaced approximately 8.1 cM apart across the human genome. Two point and multipoint linkage analyses were carried out using the variance component method. RESULTS: The strongest linkage signal was obtained on Xq27 with two point LOD scores of 4.30 for wrist BMD, and 2.57 for hip BMD, respectively. Another important region was 11q23, which achieved a maximum LOD score of 3.13 for spine BMD in multipoint analyses, confirming the results on this region in two earlier independent studies. Suggestive linkage evidence was also found on 7p14 and 20p12. CONCLUSIONS: Together with the findings from other studies, the current study has further delineated the genetic basis of bone mass and highlights the importance of increasing sample size to confirm linkage findings and to identify new regions of linkage.

A second-stage genome scan for QTLs influencing BMD variation.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture. To identify genomic regions harboring quantitative trait loci (QTLs) contributing to BMD variation, we performed a two-stage genome screen. The first stage involved genotyping of a sample of 53 pedigrees with 630 individuals using 400 microsatellite markers spaced at approximately 10-cM intervals throughout the genome. Ten genomic regions with multi- and/or two-point LOD scores greater than 1.5 were observed. In the present second-stage study, 60 microsatellite markers, with a mean spacing of about 5 cM, were genotyped in these regions in an expanded sample of 79 pedigrees that contained 1816 subjects. Each pedigree was ascertained through a proband with extreme BMD at the hip or spine. BMD at the spine (L1-4), hip (the femoral neck, trochanter, and intertrochanteric region), and wrist (the ultradistal region) was measured by dual-energy X-ray absorptiometry (DXA) and was adjusted for age, sex, height, and weight. Two-point and multipoint linkage analyses were performed for each BMD site using statistical genetic methods that are implemented in the computer package SOLAR. Several regions (7q11, 10q26, 12q13, and 12q24) achieved LOD scores in excess of 1 in the second-stage followup study. The current results replicate some of our previous linkage findings and also highlight some of the difficulties facing microsatellite linkage mapping for complex human diseases.

Several genomic regions potentially containing QTLs for bone size variation were identified in a whole-genome linkage scan.

Bone size is an important determinant of osteoporotic fractures. For a sample of 53 pedigrees that contains more than 10,000 relative pairs informative for linkage analyses, we performed a whole-genome linkage scan using 380 microsatellite markers to identify genomic regions that may contain QTLs of bone size (two dimensional measurement by dual energy X-ray absorptiometry). We conducted two- and multi-point linkage analyses. Several potentially important genomic regions were identified. For example, the genomic region 17q23 may contain a QTL for wrist (ultra distal) bone size variation; a LOD score of 3.98 is achieved at D17S787 in two-point analyses and a maximum LOD score (MLS) of 3.01 is achieved in multi-point analyses in 17q23. 19p13 may contain a QTL for hip bone size variation; a LOD score of 1.99 is achieved at D19S226 in two-point analyses and a MLS of 2.83 is achieved in 19p13 in multi-point analyses. The genomic region identified on chromosome 17 for wrist bone size seems to be consistent with that identified for femur head width variation in an earlier whole-genome scan study. The genomic regions identified in this study and an earlier investigation on one-dimensional bone size measurement by radiography are compared. The two studies may form a basis for further exploration with larger samples and/or denser markers for confirmation and fine mapping studies to eventually identify major functional genes and the associated etiology for osteoporosis.

A whole-genome linkage scan suggests several genomic regions potentially containing quantitative trait Loci for osteoporosis.

Osteoporosis is an important health problem, particularly in the elderly women. Bone mineral density (BMD) is a major determinant of osteoporosis. For a sample of 53 pedigrees that contain 1249 sibling pairs, 1098 grandparent-grandchildren pairs, and 2589 first cousin pairs, we performed a whole- genome linkage scan using 380 microsatellite markers to identify genomic regions that may contain quantitative trait loci (QTL) of BMD. Each pedigree was ascertained through a proband with BMD values belonging to the bottom 10% of the population. We conducted two-point and multipoint linkage analyses. Several potentially important genomic regions were suggested. For example, the genomic region near the marker D10S1651 may contain a QTL for hip BMD variation (with two-point analysis LOD score of 1.97 and multipoint analysis LOD score of 2.29). The genomic regions near the markers D4S413 and D12S1723 may contain QTLs for spine BMD variation (with two-point analysis LOD score of 2.12 and 2.17 and multipoint analysis LOD score of 3.08 and 2.96, respectively). The genomic regions identified in this and some earlier reports are compared for exploration in extension studies with larger samples and/or denser markers for confirmation and fine mapping to eventually identify major functional genes involved in osteoporosis.

A whole-genome linkage scan suggests several genomic regions potentially containing QTLs underlying the variation of stature.

Human height is a complex trait under the control of both genetic and environment factors. In order to identify genomic regions underlying the variation of stature, we performed a whole-genome linkage analysis on a sample of 53 human pedigrees containing 1,249 sib pairs, 1,098 grandparent-grandchildren pairs, 1,993 avuncular pairs, and 1,172 first-cousin pairs. Several genomic regions were suggested by our study to be linked with human height variation. These regions include 5q31 at 144 cM from pter on chromosome 5 (with a maximum LOD score of 2.14 in multipoint linkage analyses), Xp22 at the marker DXS1060, and Xq25 at DXS1001 on the X chromosome (with LOD scores of 1.95 and 1.91, respectively, in two-point linkage analyses). Noticeably, Xp22 happens to be the very region where a newly identified gene underlying idiopathic short stature, SHOX, maps. Based on our findings, further confirmation and fine-mapping studies are to be pursued on expanded samples and/or with denser markers for eventual identification of major functional genes involved in human height variation.

Evidence for a major gene for bone mineral density/content in human pedigrees identified via probands with extreme bone mineral density.

Bone mineral content (BMC) and/or bone mineral density (BMD, i.e. BMC scaled by bone size) are major determinants for osteoporosis, which is a serious health problem. The major determinant of variation in BMD/BMC is genetic. The few studies now available are inconsistent in the identification and/or even in the existence of major gene(s) for BMD/BMC. In 51 human pedigrees with 941 individuals (526 measured for phenotypes) identified via probands with extreme BMD values, we performed complex segregation analyses to test the existence of a genetic locus with a major effect on BMD/BMC variation. We analyzed BMD and BMC at the spine, hip and wrist jointly by employing, as the study phenotype, factor scores (FS) of the principle component that explains approximately 75% of the total BMD/BMC variation at the three sites. The results indicate that a major gene exists with a codominant effect that is responsible for approximately 16% of the FS variation when adjusted for significant effects of sex, body weight and age. A significant genotype-x-sex-x-age interaction was found, which may explain approximately 14% of the FS variation after adjusting for body weight. Testing of various models did not provide support for shared familial environmental effects but suggested the existence of residual polygenic effects, which may explain approximately 50% of the FS variation when adjusting for sex, body weight and age. This study indicates a promising aspect of studies to identify a major gene for BMD/BMC variation in our pedigrees identified via extreme probands.

A genomewide linkage scan for quantitative-trait loci for obesity phenotypes.

Obesity is an increasingly serious health problem in the world. Body mass index (BMI), percentage fat mass, and body fat mass are important indices of obesity. For a sample of pedigrees that contains >10,000 relative pairs (including 1,249 sib pairs) that are useful for linkage analyses, we performed a whole-genome linkage scan, using 380 microsatellite markers to identify genomic regions that may contain quantitative-trait loci (QTLs) for obesity. Each pedigree was ascertained through a proband who has extremely low bone mass, which translates into a low BMI. A major QTL for BMI was identified on 2q14 near the marker D2S347 with a LOD score of 4.04 in two-point analysis and a maximum LOD score (MLS) of 4.44 in multipoint analysis. The genomic region near 2q14 also achieved an MLS >2.0 for percentage of fat mass and body fat mass. For the putative QTL on 2q14, as much as 28.2% of BMI variation (after adjustment for age and sex) may be attributable to this locus. In addition, several other genomic regions that may contain obesity-related QTLs are suggested. For example, 1p36 near the marker D1S468 may contain a QTL for BMI variation, with a LOD score of 2.75 in two-point analysis and an MLS of 2.09 in multipoint analysis. The genomic regions identified in this and earlier reports are compared for further exploration in extension studies that use larger samples and/or denser markers for confirmation and fine-mapping studies, to eventually identify major functional genes involved in obesity.

Characterization of genetic and lifestyle factors for determining variation in body mass index, fat mass, percentage of fat mass, and lean mass.

In this study, we simultaneously characterized genetic and lifestyle factors (exercise, smoking, and alcohol consumption) in determining variation in body mass index (BMI), fat mass, percentage of fat mass (PFM), and lean mass while adjusting for the effects of age and sex. Six hundred fifty-eight Caucasian individuals from 48 pedigrees were studied for BMI. Among these individuals, 289 from 38 pedigrees were studied for fat mass, PFM, and lean mass measured by dual X-ray absorptiometry (DXA). After adjusting for age, sex, and lifestyle factors, the heritabilities (h(2)) of BMI, fat mass, PFM, and lean mass ranged from 0.52 to 0.57 with associated standard errors ranging from 0.09 to 0.14. After accounting for significant sex and age effects, exercise had significant effects for all the phenotypes studied, and the effects of smoking and alcohol consumption were not significant. Therefore, significant proportions of variation in BMI, fat mass, PFM, and lean mass were under genetic control, and exercise had a significant effect in reducing BMI, fat mass, and PFM and in increasing lean mass. This study warrants further genetic linkage analyses to search for genes for the obesity-related phenotypes measured by DXA in our population.

Is population bone mineral density variation linked to the marker D11S987 on chromosome 11q12-13?

Our purpose is to test linkage of human chromosome 11q12-13 to BMD variation. Chromosome 11q12-13 has been linked to three BMD-related phenotypes that are inherited as Mendelian traits in human pedigrees: an autosomal dominant high bone mass trait, autosomal recessive osteoporosis pseudoglioma, and autosomal recessive osteopetrosis. A sibling pair study with 374 sibships showed significant linkage of D11S987 to normal BMD variation, with a maximum logarithm of odds score of 3.5. However, a subsequent linkage study with a total of 595 sibling pairs demonstrated reduced significance for linkage of D11S987 to bone mineral density variation, with a logarithm of odds score less than 2.2. We genotyped five markers in a genomic region of approximately 27 cM centering on D11S987 and measured bone mineral density and other traits (weight, etc.) for 635 individuals from 53 human pedigrees. Each of these pedigrees was ascertained through a proband with bone mineral density Z-scores less than -1.28 at the hip or spine. Adjusting for age, sex, and weight as covariates, we performed two-point and multipoint linkage analyses using the variance component linkage analysis method implemented in Sequential Oligogenic Linkage Analysis Routines. We found little evidence of linkage of these five markers to bone mineral density at the spine, hip, wrist and total body bone mineral content. The maximum logarithm of odds score at these five markers was 0.25, and the maximum logarithm of odds score at D11S987 was 0.15. Therefore, although we cannot exclude the linkage of D11S987 region to bone mineral density variation, there is no evidence for linkage of the marker D11S987 on human chromosome 11q12-13 to bone mineral density variation in our study population.

Hormones, weight change and menopause.

OBJECTIVE: To determine total body weight change occurring in women at mid-life, specifically with respect to occurrence of menopause and use of estrogen. DESIGN: Retrospective analysis of body weight measurements accumulated in two cohorts of healthy women participating in studies of skeletal metabolism. SUBJECTS: Cohort 1: 191 healthy nuns enrolled in a prospective study of osteoporosis risk, aged 35-45 in 1967; cohort 2: 75 women aged 46 or older and still menstruating, enrolled in 1988 in a study of bone cell dynamics across menopause. Roughly one-third of each group received hormone replacement after menopause. MEASUREMENTS: Body weight and height, age, menstrual status and use of estrogen replacement. Cohort 1: 608 measurements at 5 y intervals spanning a period from 17 y before to 22 y after menopause; cohort 2: 1180 measurements at 6-month intervals spanning a period from 5 y prior to 5 y after menopause. RESULTS: In cohort 1 weight rose as a linear function of age (both chronological and menopausal), both before and after cessation of ovarian function, at a rate of approximately 0.43% y(-1). Neither the menopausal transition nor the use of estrogen had an appreciable effect on this rate of gain. In cohort 2 the rate of gain seemed to diminish slightly at menopause. As with cohort 1, hormone replacement (or its absence) had no appreciable effect on weight. CONCLUSIONS: The long-term, total body weight trajectory at mid-life is not influenced appreciably by either cessation of ovarian function or by hormone replacement.

Identification and in vivo characterization of the Epiphyas postvittana nucleopolyhedrovirus Ecdysteroid UDP-glucosyltransferase.

The genome of Epiphyas postvittana Nucleopolyhedrovirus (EppoMNPV) contains an ecdysteroid UDP-glucosyltransferase (egt) gene. The egt gene was completely sequenced and surrounding open reading frames identified. EppoMNPV egt is 1479 nucleotides in length encoding a protein with a predicted molecular mass of 55 kDa. Analysis of upstream sequence revealed dual TATA boxes and two CGT upstream activating region motifs. Mapping of the 5' terminus of the egt transcript identified a major transcript produced from an adenine residue 29 nucleotides downstream from the distal TATA box. No transcript was detected from a late promoter motif (GTAAG). Characterization of egt transcripts showed that poly-adenylation occurs at the 3' terminus. EppoMNPV egt transcripts were first detected in infected Epiphyas postvittana larvae by Northern hybridization at 9 h post-infection (hpi) and EGT enzyme activity was detected at 9 hpi in haemolymph from infected larvae. EppoMNPV EGT can conjugate the sugars from both UDP-glucose and UDP-galactose to ecdysone in vitro. Localization assays performed using radiolabelled ecdysone demonstrated that the conjugation of glucose or galactose, from the respective UDP-sugar, led to the prevention of the uptake of ecdysone by SF-21 cells. We propose that EGT functions to prevent the uptake of ecdysone or 20-hydroxyecdysone by metabolite and target cells respectively.

Chemistry of the diazeniumdiolates. 3. Photoreactivity.

We have found O(2)-substituted diazeniumdiolates, compounds of structure R(2)N-N(O)=NOR' that are under development for various possible pharmaceutical uses, to be rather photosensitive. With R = ethyl and R' = methyl, benzyl, or 2-nitrobenzyl, the observed product distributions suggest that two primary pathways are operative. A minor pathway involves the extrusion of nitrous oxide (N(2)O) with simultaneous generation of R(2)N(*) and R'O(*), which may then form amines, aldehydes, and alcohols. The major reaction pathway is an interesting photochemical cleavage of the N=N bond to form a nitrosamine (R(2)NN=O) and an oxygen-substituted nitrene (R'ON). The intermediacy of the O-nitrene was inferred from the production of abundant oxime, via rearrangement of the O-nitrene to a C-nitroso compound (R'ON --> O=NR'), and subsequent tautomerization to the more stable oxime. Involvement of the O-nitrene was confirmed by trapping with 2,3-dimethyl-2-butene to form the aziridine and with oxygen to generate the nitrate ester. 2-Nitro substitution on the benzyl derivative had surprisingly little effect on the reaction course. For each compound examined, minor amounts of nitric oxide (NO), presumably produced by secondary photolysis of the nitrosamine, were observed. Time-resolved infrared experiments provided additional support for the above reaction pathways and confirmed that the nitrosamine is a primary photoproduct. We have also found that the relative contributions of the reaction pathways can be altered in certain derivatives. For example, when R' = 2,4-dinitrophenyl, the contribution of the nitrosamine/O-nitrene-forming pathway was diminished. Pharmacological implications of these results are discussed.

Chemistry of the diazeniumdiolates. 2. Kinetics and mechanism of dissociation to nitric oxide in aqueous solution.

Diazeniumdiolate ions of structure R(2)N[N(O)NO](-) (1) are of pharmacological interest because they spontaneously generate the natural bioregulatory species, nitric oxide (NO), when dissolved in aqueous media. Here we report the kinetic details for four representative reactivity patterns: (a) straightforward dissociation of the otherwise unfunctionalized diethylamine derivative 2 (anion 1, where R = Et) to diethylamine and NO; (b) results for the zwitterionic piperazin-1-yl analogue 4, for which the protonation state of the neighboring basic amine site is an important determinant of dissociation rate; (c) data for 5, a diazeniumdiolate derived from the polyamine spermine, whose complex rate equation can include terms for a variety of medium effects; and (d) the outcome for triamine 6 (R = CH(2)CH(2)NH(3)(+)), the most stable structure 1 ion identified to date. All of these dissociations are acid-catalyzed, with equilibrium protonation of the substrate preceding release of NO. Specific rate constants and pK(a) values for 2-6 have been determined from pH/rate profiles. Additionally, a hypsochromic shift (from approximately 250 to approximately 230 nm) was observed on acidifying these ions, allowing determination of a separate pK(a) for each substrate. For 6, the pK(a) value obtained kinetically was 2-3 pK(a) units higher than the value obtained from the spectral shift. Comparison of the ultraviolet spectra for 6 at various pH values with those for O- and N-alkylated diazeniumdiolates suggests that protonation at the R(2)N nitrogen initiates dissociation to NO at physiological pH, with a second protonation (at oxygen) accounting for both the spectral change and the enhanced dissociation rate at pH <4. Our results help to explain the previously noted variability in dissociation rate of 5, whose half-life we found to increase by an order of magnitude when its concentration was raised from near-zero to 1 mM, and provide mechanistic insight into the factors that govern dissociation rates among diazeniumdiolates of importance as pharmacologic progenitors of NO.

The secondary amine/nitric oxide complex ion R(2)N[N(O)NO](-) as nucleophile and leaving group in S9N)Ar reactions.

Ions of structure R(2)N[N(O)NO](-) and their alkylation products have seen increasing use as nitric oxide (NO)-generating agents for biomedical research applications. Here we show that such diazeniumdiolate anions can readily displace halide from a variety of electrophilic aza- or nitroaromatic substrates to form O(2)-arylated derivatives of structure R(2)N-N(O)=N-OAr. The site of arylation and the cis arrangement of the oxygens were confirmed by X-ray crystallography. Displacement by various nucleophiles showed R(2)N[N(O)NO](-) to be a reasonably good leaving group, with rate constants for displacement by hydroxide, methoxide, and isopropylamine that were between those of chloride and fluoride in the S(N)Ar reactions we surveyed. The Meisenheimer intermediate could be spectrally observed. These O(2)-aryl diazeniumdiolates proved capable of reacting with the nucleophilic sulfur of the HIV-1 p7 nucleocapsid protein's zinc finger assembly to eject the zinc, disrupting a structural motif critical to viral replication and suggesting possible utility in the drug discovery realm.