Pheochromocytoma in von Hippel-Lindau disease: clinical presentation and mutation analysis in a large, multigenerational kindred.

The clinical presentation and characterization of the mutation in members of a large kindred with von Hippel-Lindau disease (VHLD) and pheochromocytoma were examined. Twenty-five proven cases of VHLD occurring in four generations of a large kindred have been followed since 1964, and pheochromocytoma has occurred in 17. Symptoms of pheochromocytoma developed at an early age, on average at 12.5 +/- 1.3 yr, and definitive diagnosis and treatment of pheochromocytoma occurred at 19.9 +/- 2.6 yr. Significantly higher urine catecholamine concentrations were observed in younger patients than in older ones. Mutation analysis was performed in 14 family members, and a new mutation in the VHLD gene was identified in 11; this mutation is a G to T change at nucleotide 658 that results in the substitution of a serine for an alanine residue at position 149 of the polypeptide chain. Seven of the 11 patients with the mutation have VHLD; four, all 10 yr old or less, are asymptomatic and have no evidence of disease, but are at high risk for developing VHLD. These children are being followed closely for clinical and biochemical manifestations. The characterization of this new mutation has permitted identification of family members who are likely to develop VHLD.

Osteopenia, pathological fractures, and increased urinary calcium excretion in schizophrenic patients with polydipsia.

Ten male chronic schizophrenic patients with polydipsia and 10 nonpolydipsic controls, matched for gender, diagnosis, duration of illness, age, and race, were studied by dual- and single-photon absorptiometry to estimate bone density of the lumbar spine and radius and by 24-hr urine collections to estimate urinary electrolyte excretion. Bone density was normal in the control group, but was abnormally low in the polydipsic group, which had a markedly increased incidence of fractures. Electrolyte excretion was normal in the control group and in the polydipsic group when water intake was restricted to normal amounts; increased urinary sodium and calcium excretion occurred in proportion to polydipsia. As polydipsia is associated with a number of physiological changes, the cause of the osteopenia is unclear; we suggest that a negative calcium balance caused by increased urinary calcium excretion induced by extracellular space expansion may play an important role in the causation of the skeletal changes.

Thyroid function and response to 48-hour sleep deprivation in treatment-resistant depressed patients.

BACKGROUND: Clinical depression is associated with abnormalities of the hypothalamic-pituitary-thyroid axis. Changes in thyroid function during sleep deprivation may be related to its antidepressant effects. METHODS: Levels of thyroid-stimulating hormone, tri-iodothyronine, tri-iodothyronine uptake, thyroxine, and free thyroxine were measured before, during, and after a 48-hour sleep deprivation in nine treatment-resistant depressed patients. Clinical state was assessed every 4 hours. A retrospective study of 26 similar patients was added for cross-validation. RESULTS: Significant increases in thyroid-stimulating hormone and tri-iodothyronine during sleep deprivation were not correlated with clinical improvement. Sleep deprivation responders had lower tri-iodothyronine uptake levels than nonresponders in both the prospective (p <.02) and the retrospective (p <.03) samples. CONCLUSIONS: The lower tri-iodothyronine uptake values in responders may identify a subgroup of depressed patients who respond to sleep deprivation by virtue of some abnormality of the hypothalamic-pituitary-thyroid axis that is temporarily corrected by sleep deprivation.

Age-related morphine kinetics.

The kinetic disposition of morphine was compared in 13 young and 7 older healthy subjects after a single intravenous dose of 10 mg/70 kg morphine. The apparent volume of distribution at steady state in elderly subjects was only half of that in young subjects. This difference was derived from reductions in both central and peripheral kinetic compartment volumes in the older subjects. The beta elimination phase for morphine was more rapid, but its plasma clearance was reduced in older subjects. Calculation of the peripheral compartment morphine concentration for these subjects indicated that drug concentration in this kinetic space was higher in older subjects for 1.5 hr after dosing.

Effects of age and disease on the pharmacokinetics and pharmacodynamics of sulindac.

The disposition and effect on hemostasis of a single 150 mg dose of sulindac was studied in young healthy subjects and in older patients with arthritis. Older patients were restudied after 2 weeks of sulindac, 150 mg b.i.d. The only difference in disposition of the first dose was a reduced plasma sulfone metabolite concentration in the elderly patients with arthritis. Chronic sulindac dosing resulted in accumulation of the drug and its sulfone and sulfide metabolites in plasma to a greater extent than previously reported for young subjects. No differences in renal clearance of sulindac and its sulfone metabolite related to age or chronic drug dosing were observed. No renal excretion of the active sulfide metabolite was detected. Bleeding time in the elderly patients was shorter than in the young healthy subjects before sulindac dosing, but was prolonged in the elderly patients after 2 weeks of dosing to values similar to control data from the young healthy subjects. This change correlated weakly with plasma sulfide metabolite concentrations. Differences in bleeding time were not reflected in changes in platelet aggregation induced by adenosine diphosphate either with respect to age or chronic drug dosing. Our data provide no justification for lowering the recommended dose of sulindac for patients older than 65 years of age.

Physiology of the menstrual cycle.

Modern techniques of bioassay have permitted correlation of hormonal secretion with genital tissue changes during the normal menstrual cycle. During the follicular phase, estrogen secretion rises while other hormone levels are low. At ovulation luteinizing hormone and follicle-stimulating hormone surges are associated with falling estrogen levels. Secretions of progesterone and estrogen again are characteristic of the luteal phase ending with menstruation. Gonadotrophin-releasing hormones are detectable just before the luteinizing hormone and follicle-stimulating hormone surges. Basal body temperature rises with ovulation and is still the most reliable clinical indicator, although ferning and spinnbarkeit (when present) are also quite helpful. Vaginal smears are probably less useful except in the hands of experienced observers.

PIP: The correlation of hormonal secretion with genital tissue changes during the normal menstrual cycle has been made possible employing modern techniques of bioassay. Estrogen secretion rises and other hormone levels are low buring the follicular phase. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) surges are associated with decreasing estrogen levels at ovulation. The luteal phase ending with menstruation is characterized by the secretion of progesterone and estrogen. Prior to LH and FSH surges gonadotrophin-releasing hormones are detectable. The rise of basal body temperature with ovulation is the most reliable indicator of ovulation although ferning and spinnbarkeit are also useful. Vaginal smears may be of use when handled by experienced people.

Successful in vitro antigen-dependent activation of 24-hour-old peripheral blood lymphocytes.

We describe a simple, rapid and reproducible in vitro culture system in which human peripheral blood lymphocytes (PBLs), donated 24 h prior to initiation of culture can be stimulated to produce antigen-specific antibodies. Peripheral blood lymphocytes purified by Ficoll-Hypaque centrifugation were passed over a G10 Sephadex column and then activated in vitro in the presence of 0.003% staphylococcus Cowan A, 2.8 x 10(-6) M indomethacin and appropriate concentrations of tetanus toxoid antigen. After the first 24 h in culture, a five-fold concentrated supernatant from an allogeneic mixed lymphocyte culture was added. The cell surface phenotypes of the PBLs were analyzed by flow cytometry at the initiation and termination of culture, in order to provide a comprehensive characterization of the cellular composition of a successful in vitro stimulation system. Our results clearly show that the majority of peripheral blood B cells can be induced to an activated stage (blast transformation) and interleukin 2 (IL-2) receptor expression, following very simple manipulations of the lymphoid population. Tetanus toxoid-specific antibody production can be readily generated in this cell population. In contrast, T cells were not activated to express IL-2 receptors and reach blast transformation, and did not show appreciable proliferation. Our system provides a population of B cells producing antibodies of desired specificity which could be utilized for the generation of human hybridomas or could serve as a donor population for antibody engineering via the combinatorial library approach. Careful light scattering and cell surface phenotypic analyses of the cells entering, proliferating and differentiating in these cultures enabled several novel observations to be made.

Fluorescein-specific hybridomas derived from primary mice exhibit more stringent growth requirements than do hybrids from pre-immune animals.

Although the generation of antigen-specific hybridoma cell lines from animals which have been multiply immunized is now a routine procedure, the derivation of hybridomas following a single in vivo antigen injection has proven to be much more difficult to accomplish. We show that the addition of an interleukin-containing supernatant derived from rat spleen cells which have been stimulated with concanavalin A (ConA SN) to hybrids after the initial cloning step results in the consistently successful isolation of IgM anti-fluorescein specific hybridomas. However, addition of the same supernatant to fused cultures simultaneously with the addition of the HAT selection medium results in the loss growing cells. In contrast to the situation with primary hybridomas, the growth of secondary hybridomas is inhibited by the addition of ConA SN at the cloning step. Following successful cloning, there is a time-dependent variation in the sensitivity of all cell lines to ConA SN.

Xenobiotic mediated inhibition of hepatic uroporphyrinogen decarboxylase activity in 17-day-old chick embryo liver cells in culture.

Uroporphyrin, heptacarboxylic acid porphyrin and coproporphyrin were the major porphyrins to accumulate when phenobarbital, nifedipine, 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) and 3,3',4,4'-tetrachorobiphenyl (TCBP) were added to chick embryo hepatocyte culture. This pattern of porphyrin accumulation is consistent with the demonstration that these chemicals inhibit uroporphyrinogen decarboxylase (UROG-D). The degree of UROG-D inhibition observed was: TCBP (39%), Ox-DDC (39%), nifedipine (25%) and phenobarbital (50%). Since significant UROG-D inhibition was observed when the bulk of the porphyrins in the crude enzyme preparation was removed by gel filtration, it is unlikely that porphyrins produce the enzyme inhibition. When succinylacetone, a potent inhibitor of delta-aminolevulinic acid dehydratase, was coadministered with Ox-DDC, phenobarbital, TCBP and nifedipine, UROG-D inhibition was not observed. These results suggest that heme biosynthesis must proceed in order for xenobiotic mediated UROG-D inhibition to occur.

Measurement of methaemalbumin in plasma.

Using the method of Chong and Owen (1967), the normal range of methaemalbumin in plasma was 0 to 0.6 mg/100 ml, expressed as milligrams of haematin per cent. Previous results, using the method of Shinowara and Walters (1963), reported a normal range of 0 to 8.0 mg/100 ml, but it was expressed as milligrams of haemoglobin percent. The conversion factor from the Shinowara method is as follows: mg haematin % = mg haemoglobin % x 0.04.

Prevalence and significance of tobacco smoking in impotence.

We investigated the incidence of cigarette smoking in a sample of patients and compared these figures with estimates of smoking among males in the general population. Among 178 impotent patients the number of current smokers (58.4%) and current ex-smokers combined (81%) was significantly higher than would be expected among males in the general population. In each age group, and at all levels of tobacco use, impotent patients smoked more than would be expected from population estimates. Smoking and nonsmoking impotent patients did not differ in terms of their hormonal profile; however, mean penile blood pressure (PBI) was lower among patients who smoked than among those who did not. A significantly higher proportion (20.9%) of impotent patients with a history of smoking showed abnormally low PBI compared with nonsmoking patients (8.8%). This study adds to preliminary evidence that smoking may be a significant risk factor in impotence, and its effects are evident in the small vasculature.

Reliable fifteen-minute assay for theophylline.

We describe a reliable, rapid high pressure liquid chromatography assay for serum theophylline which can be performed on 0.5 ml or smaller samples. The procedure involves extraction, centrifugation and sampling all in the same disposable centrifuge tube. In the therapeutic range the coefficients of variation for within-day and day-to-day assays are 1.0% and 2.6%, respectively. The sensitivity of the method is better than 1 microgram/ml in serum. The minimum number of steps, rapid elution and use of only peak heights contribute to the reliability and rapidity of the assay.

Studies of nonoxynol-9. I. The effect on the vaginas of rabbits and rats.

A standard rabbit vaginal irritation test was used to evaluate the morphologic reaction to an aqueous solution of a nonionic spermicidal detergent, nonoxynol-9 (N-9). Various amounts of N-9, between 2.5 mg and 50 mg, were incorporated into cylindrical collagen sponges which were retained in the vagina for 10 days. Changes in the continuity of the epithelial lining, edema of the submucosal layer, and the inflammatory cell infiltrate of this layer were proportional to the amount of N-9 tested. An increase in semiquantitative score for the inflammatory changes in the vagina was noted for increasing amounts of N-9. In young adult female rats treated vaginally with 5 mg/100 gm body weight/day of aqueous N-9 for 5 to 20 days, the number of cells (DNA) was significantly increased on day 5. The collagen content in the vaginal wall (hydroxyproline) was significantly increased after 15 and 20 daily injections of the detergent. We conclude that both morphologic and chemical changes in the vagina are compatible with the inflammatory reaction induced by the detergent tested.

Calibration drift in the automated determination of calcium and magnesium by atomic absorption.

Calibration drift was studied in the automated on-line determination of calcium and magnesium by atomic absorption over periods of up to four hours. Automatic correlation for drift improved the precision of results.

A comprehensive laboratory computer system working round the clock.

The special features of a laboratory computer system operated by laboratory staff around the clock are presented. The system provides registration of work, production of worksheets, on-line data acquisition and reporting results. The reasons for the special design features are discussed.

Moving from policy to practice in curriculum change at the University of Virginia School of Medicine, East Carolina University School of Medicine, and SUNY-Buffalo School of Medicine.

A set of formative evaluation studies from the medical schools of the University of Virginia (UVA), East Carolina University (ECU), and the State University of New York at Buffalo (SUNY-Buffalo) portrays, in qualitative and quantitative terms, evidence of achievements and obstacles to the curricular reform supported by The Robert Wood Johnson Foundation's Generalist Physician Initiative (GPI). In this paper, innovations in the under-graduate curriculum, a specific course, and instructional strategies are examined. Individual interviews of faculty and focus groups with students assessed opinions about curricular change at the University of Virginia. Questionnaires and focus groups provided information about the impact of course changes at East Carolina University. Questionnaires completed by students provided information of the effect of modifying the instructional strategies at SUNY-Buffalo. The obstacles to implementing change at the three schools included breakdowns in the faculty's understanding and support of change, lack of skills required to implement change, and weakness in coordinating and assessing planned change. Although the GPI catalyzed changes in the content and conduct of generalist education at the three schools, many lessons were learned that are applicable to other medical schools.

Changes in the fatty acid profile of lung, liver and serum of rats intratracheally given silica.

The esterified (E) and nonesterified (NE) fatty acid level and profile in the lung, serum, and liver of rats are significantly altered after intratracheal administration of silica. The changes include a silica-specific increase of the total long chain (C16-C20:4) fatty acid content in the lung, and a decrease in the serum and liver of both groups of rats intratracheally given silica and/or saline. In the silicotic lung, arachidonate and palmitate accumulated at the highest rate. A heat-labile, high-molecular weight component from lung homogenates increases lipogenesis in isolated hepatocytes in vitro. These findings, taken together with evidence indicating increased lipogenesis in the liver of rats treated with silica under identical conditions, suggest a lung-liver communication mechanism which coordinates lipid uptake by the lung and lipid synthesis and release by the liver. The stimulatory factor identified in lung homogenates might play an important regulatory role-for hepatic lipogenesis in rats developing silicotic lungs.

Dealing with specimen problems in a computerised clinical chemistry laboratory.

The use of a laboratory computer system to deal with specimen problems is described. Problems such as 'insufficient material' or 'haemolysis' are entered by reception staff or by bench workers so that resolution of the problem will not be overlooked; problems are then listed and dealt with by the duty biochemist. Specimens on printed worksheets are annotated 'urgent' where appropriate. Results obtained on-line, which are above top standard, automatically cause the specimen to reappear on the next worksheet marked 'dilution required'. Data relating to its use over a three-month period are presented together with some comments on the causation of specimen problems and their prevention.

Use of an enzyme standard in the measurement of enzymes in a Clinicon reaction rate analyser.

Measurement of enzyme activity in a Clinicon reaction rate analyser was carried out in two ways: one method was based on absolute calibration and the other used an enzyme standard which involved adjustment of results according to the value for the standard obtained in each batch. Batch calibration significantly improved long-term precision and reduced differences between results obtained in different instruments.

A comparison of five curve-fitting procedures in radioimmunoassay.

Data obtained from routine analytical radioimmunoassays were processed using five curve-fitting procedures, viz. 'Amersham', single binding site, four parameter logistic, a linear logit-log and a polynomial logit-log. The polynomial logit-log procedure gave the best fit, but this was probably due to the inherent flexibility of this curve-fitting process since the analytical precision achieved with it was not better than what was obtained with most of the other procedures. A limited study failed to show that statistical weighting of data before curve fitting had any practical advantage.