Association of fractures with caffeine and alcohol in postmenopausal women: the Iowa Women's Health Study.

OBJECTIVE: To assess whether alcoholic and caffeinated beverages are associated with risk of fractures in women. SETTING: Population-based sample surveyed by post. SUBJECTS: A total of 34 703 postmenopausal Iowan women aged 55-69 years were surveyed. DESIGN: A cohort of women reported alcoholic and caffeinated beverage intake and were followed for 6.5 years for fracture occurrence. Relative risks (RR) and 95% confidence intervals (CI) were computed using Cox proportional hazards regression. Covariates included age, tobacco use, physical activity, body mass index (BMI), waist to hip ratio (WHR), oestrogen use and calcium intake. RESULTS: At least one fracture was reported by 4378 women (389 upper arm, 288 forearm, 1128 wrist, 275 hip, 416 vertebral and 2920 other fractures). The adjusted RR for highest versus lowest caffeine intake quintiles was 1.09 (95% CI 0.99-1.21) for combined fracture sites. Wrist fractures were associated positively (RR for extreme quintiles 1.37, 95% CI 1.11-1.69) and upper arm fractures were negatively associated (RR 0.67, 95% CI 0.48-0.94) with caffeine intake. The age-adjusted RR of total fractures for highest versus lowest frequency of beer usage was 1.55 (95% CI 1.25-1.92) and for liquor was 1.25 (95% CI 1.03-1.54). No other association was found between any specific fracture site and alcohol intake. CONCLUSIONS: We found a modest increase in fracture risk associated with highest caffeine intake, varying by site. Alcohol intake was low, but it also showed a weak positive association with fracture risk.

Isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes, exemplified by purification of glucoamylase.

A highly purified preparation of glucoamylase G1 from Aspergillus niger was found, by isoelectric focusing in immobilized pH gradients, to contain a major form with a pI of 3.50 and a number of minor, more acidic and more basic contaminants. The major isoform was purified to homogeneity by recycling isoelectric focusing in a multicompartment electrolyzer, by confining this form in between two zwitterionic membranes, with pI 3.49 at the anodic side and pI 3.52 at the cathodic side. Recoveries were high (90%) and, notwithstanding the rather low operational pH, the electrosmotic flow was minimal and no protein precipitation occurred up to concentrations of 2.5 mg/ml (at the isoelectric point). The forms resolved in an analytical focusing gel were subjected to two types of in situ enzyme detections, by the glucose oxidase peroxidase (GOP) test and by the starch-iodine test. By both criteria all resolved zones exhibited enzyme activity, the GOP assay, however, following more closely the Coomassie blue stained protein profile. By computer modelling, it is shown that it is impossible to obtain linear pH gradients at such low pH values (pH 2.5-4.5 intervals) when the mixture has a low buffering power (beta = 2.0 mequiv.l-1 pH-1). When the beta power was gradually raised (beta = 4, beta = 6, beta = 8) the pH gradient became progressively linear until, in a recipe with beta = 10 mequiv.l-1 pH-1 full linearity of the pH gradient could be obtained. This is shown to be due to the substantial buffering power of bulk water in the pH 2.5-3.5 region.

Effects of glucose and insulin on development of impaired insulin action in muscle.

Rat hindquarters were perfused for 2 h with either 0, 5, or 25 mM glucose in combination with either 0, 50, or 20,000 microU insulin/ml, whereupon responsiveness of glucose uptake to 20,000 microU insulin/ml and 25 mM glucose was measured. Perfusion with 25 mM glucose and 20,000 microU insulin/ml resulted in an initial glucose uptake of 43.6 +/- 3.9 mumol.g-1.h-1, which decreased to 18.7 +/- 1.6 mumol.g-1.h-1 after 2 h (P less than 0.001). Omission of glucose from the perfusate prevented the decrease in responsiveness, whereas 5 mM glucose caused a lesser decrease (to 28.3 +/- 2.2 mumol.g-1.h-1). At 0 and 50 microU insulin/ml the effects of glucose were present but were less pronounced. The decrease in insulin responsiveness of glucose uptake (55%) was accompanied by a lesser decrease (29%) in muscle glucose transport, whereas glucose transport in muscle membrane vesicles, muscle insulin binding, and insulin receptor tyrosine kinase activity were unchanged. Muscle glycogen synthase activity decreased (P less than 0.005) during perfusion with 25 mM glucose and 20,000 microU insulin/ml but did not decrease during perfusion with no glucose and 20,000 microU insulin/ml. It is concluded that insulin responsiveness of glucose uptake in muscle is decreased by exposure to glucose in a dose-dependent manner and the inhibitory effect of glucose is enhanced by simultaneous insulin exposure. The mechanism behind this insulin resistance could partly be explained by a decrease in muscle membrane glucose transport, possibly caused by changes in intracellular milieu.

Mechanisms limiting glycogen storage in muscle during prolonged insulin stimulation.

The extent to which muscle glycogen concentrations can be increased during exposure to maximal insulin concentrations and abundant glucose was investigated in the isolated perfused rat hindquarter preparation. Perfusion for 7 h in the presence of 20,000 microU/ml insulin and 11-13 mM glucose increased muscle glycogen concentrations to maximal values 2, 3, and 3.5 times above normal fed levels in fast-twitch white, slow-twitch red, and fast-twitch red fibers, respectively. Glucose uptake decreased (mean +/- SE) from 34.9 +/- 1.2 mumol.g-1.h-1 at 0 h to 7.5 +/- 0.7 after 7 h of perfusion. During the perfusion muscle glycogen synthase activity decreased and free intracellular glucose and glucose 6-phosphate increased indicating that glucose disposal was impaired. However, glucose transport as measured by the uptake of 3-O-[14C]methyl-D-glucose was also markedly decreased after 5 and 7 h of perfusion compared with initial values. Total muscle water concentration decreased during glycogen loading of the muscles. Mechanisms limiting glycogen storage under maximal insulin stimulation include impaired insulin-stimulated membrane transport of glucose as well as impaired intracellular glucose disposal.

Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake.

The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing, impairment of insulin action on muscle glucose transport and uptake. Thus maximal insulin-stimulated glucose uptake at 12 mM-glucose decreased from 34.8 +/- 1.9 to 11.5 +/- 1.1 mumol/h per g (mean +/- S.E.M., n = 10) during 5 h perfusion. This decrease in glucose uptake was accompanied by a similar change in muscle glucose transport as measured by uptake of 3-O-[14C]-methylglucose. Simultaneously, muscle glycogen stores increased to 2-3.5 times initial values, depending on fibre type. Perfusion for 5 h in the presence of glucose but in the absence of insulin decreased subsequent insulin action on glucose uptake by 80% of the effect of glucose with insulin, but without an increase in muscle glycogen concentration. Perfusion for 5 h with insulin but without glucose, and with subsequent addition of glucose back to the perfusate, revealed glucose uptake and transport similar to initial values obtained in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.

Immunostaining of rocket immunoelectrophoresis precipitates too weak for identification following staining with Coomassie brilliant blue.

Low enzyme concentrations can be determined quantitatively using rocket immunoelectrophoresis if the resolved peaks are transferred electrophoretically to a nitrocellulose membrane and immunostained.