Human cystinosis: intracellular deposition of cystine.

Membrane-limited inclusions were found in the cytoplasm of cells of the rectal mucosa, leukocytes, and cultured fibroblasts from two humans with cystinosis. Most of the inclusions contained amorphous material, presumably cystine. In cells of the rectal mucosa the material appeared frequently crystallized. This was rarely seen in leukocytes, and never in cultured fibroblasts. The fact that acid phosphatase could be demonstrated consistently in the organelles responsible for sequestration of cystine indicates that they are lysosomes.

Mapping the locus of the H-Y gene on the human Y chromosome.

The H-Y locus is on the short arm of the human Y chromosome in most individuals but on the long arm in at least one of 17 individuals with structural abnormalities of the Y.

Ethylmalonic-adipic aciduria. In vivo and in vitro studies indicating deficiency of activities of multiple acyl-CoA dehydrogenases.

The mechanisms underlying ethylmalonic-adipic aciduria were studied in a 5-yr-old girl. Oxidation of radioactive substrates by cultured skin fibroblasts from the proband and asymptomatic family members was also determined and compared to that by normal fibroblasts and that by cells from a patient with glutaric aciduria type II. Feeding medium-chain triglycerides promptly induced vomiting and lethargy accompanied by a pronounced increase of urinary ethylmalonate. Significant increases of serum isovalerate and urinary isovalerylglycine were observed after leucine feeding, but urinary glutarate increased only slightly after lysine feeding. Thus, the results from clinical investigation remained equivocal as to whether pathways other than fatty acid oxidation were blocked in our patient. Oxidation of [1-(14)C]butyrate by cultured skin fibroblasts from the proband was reduced to 14% of control. In vitro oxidation of [2-(14)C]lysine and [2-(14)C]leucine was also reduced to 28 and 23% of control, respectively. Much more severe reduction in oxidation of these three substrates (3, 9, and 9%, respectively) was observed in glutaric aciduria type II cells. These results indicated that in the proband, degradative pathways of fatty acids, lysine, and leucine are blocked at the steps of butyryl-CoA, glutaryl-CoA, and isovaleryl-CoA dehydrogenases, respectively, as in the case of glutaric aciduria type II. Because activities of multiple acyl-CoA dehydrogenases are reduced, a deficiency of electron-transferring flavoprotein, which serves as a hydrogen-acceptor for these dehydrogenases, is postulated as the underlying mechanisms of these two diseases, but a genetic heterogeneity was indicated by significant differences in the residual activities in these two types of cells. The hypothesis of more than one mutant allele of an autosomal recessive gene was also suggested by the study on cells from asymptomatic members of the family.

Persistent testicular delta5-isomerase-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD) deficiency in the delta5-3beta-HSD form of congenital adrenal hyperplasia.

A partial testicular defect in testosterone secretion has been documented in a pubertal male with a congenital adrenal hyperplasia due to hereditary deficiency of the delta5-isomerase-3beta-hydroxysteroid dehydrogenase enzyme complex (delta5-3beta-HSD). Diagnosis of the enzymatic defect is based on the clinical picture of ambiguous genitalia and salt-losing crisis in infancy, together with high urinary delta5-pregnenetriol and plasma dehydroepiandrosterone when the patient was taken off replacement corticoid treatment. No hormonal response to ACTH or salt deprivation was demonstrable. In addition, in vivo studies revealed a partial enzymatic defect in the testis. Although plasma testosterone was low-normal (250 ng/100 ml), plasma delta5-androstenediol was markedly elevated and rose to a greater extent than testosterone after human chorionic gonadotropin administration. In vitro testicular incubation studies suggested a testicular delta5-3beta-HSD enzyme defect with less delta4 products formed from delta5 precursors than in a control testis. Histochemical studies of the testis were also consistent with this defect. Testicular biopsy revealed spermatogenic arrest, generally diminished Leydig cells, but with focal areas of Leydig cell hyperplasia as well as benign Leydig cell hyperplasia as well as benign Leudig cell nodules within the spermatic cord. In vivo studies of steroid metabolism suggested intact peripheral or hepatic delta5-3beta-HSD activity. These studies imply that delta5-3beta-HSD activity differs in the gonad, adrenal, and peripheral organs. These findings are compatible with the concept that the enzyme complex consists of subunits and/or that enzymes in these organs are under different genetic control.

Intestinal beta-galactosidases. II. Biochemical alteration in human lactase deficiency.

Despite the high prevalence of intestinal lactase deficiency in some racial groups and in patients with intestinal disease, the biochemical defect has not been characterized. In the preceding paper normal intestine was found to have two lactases with distinctly different pH optima. Therefore, pH activity curves of homogenates from lactase-deficient intestine were studied, and the pH optimum was found to be shifted from the normal of 5.8 to 4.8. Density gradient ultracentrifugation of intestinal material from five lactase-deficient patients demonstrated absence of a lactase with pH optimum 6.0 and molecular weight 280,000. A second lactase with pH optimum 4.5 and molecular weights of 156,000 and 660,000 remained at normal levels accounting for the shift in the pH optimum in whole intestinal homogenates. In addition, three of the five patients had absence of a smaller beta-galactosidase (molecular weight 80,000) that had specificity only for synthetic substrates. Although not a lactase, this enzyme had a pH optimum identical with the missing lactase, and its activity was inhibited by lactose in a partially competitive manner suggesting that it is capable of binding lactose. It is possible that this enzyme is a precursor or fragment of the missing lactase.The residual lactase activity provided by the lactase with low pH optimum represents 20-70% of the activity of the missing enzyme, and yet these patients are not able to digest dietary lactose. Thus it appears that the residual enzyme plays no significant role in the hydrolysis of ingested lactose.

Efficacy of the insulin pump in the home treatment of pregnant diabetics.

The efficacy and feasibility of the insulin infusion pump in pregnancy were examined in seven class D to FR diabetics, maintained on the pump at home from 10 to 29 wk gestation until delivery. An improvement in glucose control was achieved within the first month and sustained to term. Home monitoring demonstrated a fall in mean blood glucose levels from 135 mg/dl range 98-175, prepump) to 104 mg/dl (range 84-120) and a 25-30% reduction in standard deviations during pump treatment. Furthermore, glycosylated hemoglobin levels were normalized in each patient. During periodic inpatient evaluations, mean 24-h plasma glucose levels were slightly, but not significantly, lower after pump treatment (97 vs 86 mg/dl). However, glycemic excursions were strikingly diminished; MAGE values and standard deviations fell by 45% and 34%, respectively. The total daily insulin dose required at the start of pump treatment was 31% less than the conventional dose used before the pump (P less than 0.002). Thereafter the insulin dose increased by approximately 2.5 U/wk, with the basal infusion remaining at 40% of the total dose throughout the pregnancy. All infants were born at term, had no macrosomia or neonatal problems, and had normal intravenous glucose tolerance tests at 2 h of age. We conclude that the insulin infusion pump, managed at home, is a highly efficient way to achieve normal or near-normal glucose levels in the pregnant diabetic.

Long-term improvement of metabolic control with the insulin pump does not reverse diabetic microangiopathy.

Restoration of near-normal glucose metabolism with the insulin pump reduces retinal fluorescein leakage and microalbuminuria in diabetes. However, it is not known whether these functional changes reflect a true reversal of diabetic retinopathy or nephropathy. To evaluate this question, we studied the effect of 1-2 yr of insulin pump treatment on clinical endpoints in 17 type I diabetic patients. In each patient, plasma glucose and total glycosylated hemoglobin levels fell to normal or near-normal levels. The total daily dose of insulin given during the first month of pump treatment (52 +/- 5 U/day) was comparable to that given during conventional treatment (44 +/- 3 U/day) and varied little over the 1-2 yr period of observation. Ten eyes without retinopathy at the start of the study remained without retinopathy after 15-23 mo of pump treatment. One of eleven eyes with background retinopathy developed proliferative retinopathy and 3 of 13 eyes with proliferative retinopathy progressed during pump treatment. Similarly, no improvement in renal function was observed in the six patients with diabetic nephropathy on entry to the study. In the first month of pump treatment, proteinuria consistently fell to values 30% below prepump levels (P less than 0.005). However, the diminution in proteinuria was not sustained and all remain proteinuric after 13-18 mo of pump therapy. Serum creatinine rose slightly and creatinine clearance did not significantly change. These data suggest that insulin pump treatment does not reverse established diabetic microvascular complications, despite a sustained improvement in metabolic control for up to 2 yr.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal insulin delivery in adolescents with diabetes: impact of intensive treatment on psychosocial adjustment.

The psychosocial effects of recent advances in the management of diabetes mellitus are unknown and could conceivably be adverse, particularly during the critical period of adolescence. Seven teenagers were evaluated by standard psychosocial scales and a detailed questionnaire before and on completion of a 6-mo intensive management program utilizing home glucose monitoring and multiple injections or the insulin infusion pump. All achieved improved metabolic control with inpatient glucose values (during 24-h monitoring) falling from 244 +/- 58 to 108 +/- 10 mg/dl, glycosylated hemoglobin levels falling from 11.8 +/- 2.9% to 8.4 +/- 1.7%, and home glucose levels averaging 121 +/- 16 mg/dl. Standardized scales evaluating depression, diabetic adjustment, self-esteem, and social adjustment indicated no deterioration in psychosocial functioning. There was a statistically significant increase in locus of control scores, suggesting an improved sense of internal control of life events. The program questionnaire revealed a positive response to both the program and the control devices used. This study suggests that the positive metabolic benefits of intensive diabetic management during adolescence are not offset by adverse psychosocial effects and indeed positive psychosocial benefits may result.

Pulsatile subcutaneous nocturnal administration of GnRH by portable infusion pump in hypogonadotropic hypogonadism: initiation of gonadotropin responsiveness.

GnRH was administered subcutaneously in hourly pulses for 10 consecutive nights to two immature males with Kallmann's Syndrome using a portable, battery-operated infusion pump adapted for home use. Pulsatile GnRH produced a progressive increase in urinary gonadotropin excretion, a significant increase in mean basal plasma FSH, pulsatile LH release, and an increased LH response to a standard 3 hour GnRH infusion test. One subject developed a striking increment in plasma testosterone in response to GnRH pulses, as well as a biphasic LH response to the 3 hour infusion.

Parathyroid function and vitamin D metabolism during human growth hormone replacement.

Changes in calcium and phosphorus metabolism were studied in nine children with GH deficiency before and during human GH replacement therapy. Parathyroid function and serum concentrations of physiologically important vitamin D metabolites were examined to determine their relationship to changes in mineral metabolism. By comparison with pretreatment values, the GH-treated children showed significant increases in growth rate and renal tubular phosphate reabsorption and a significant decrease in urinary calcium excretion after a standardized oral load. There was no significant change in serum concentrations of parathyroid hormone, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, or 24,25-dihydroxyvitamin D or in nephrogenous cAMP excretion. Although GH has been proposed as a regulator of vitamin D metabolism, the present study demonstrates that the anabolic changes in calcium and phosphorus metabolism accompanying GH therapy are not mediated via changes in parathyroid hormone or vitamin D status.

X-linked adrenal hypoplasia congenita: a mutation in DAX1 expands the phenotypic spectrum in males and females.

X-linked adrenal hypoplasia congenita (AHC) is a disorder associated with primary adrenal insufficiency and hypogonadotropic hypogonadism (HH). The gene responsible for X-linked AHC, DAX1, encodes a member of the nuclear hormone receptor superfamily. We studied an extended kindred with AHC and HH in which two males (the proband and his nephew) were affected with a nucleotide deletion (501delA). The proband's mother, sister, and niece were heterozygous for this frameshift mutation. At age 27 yr, after 7 yr of low dose hCG therapy, the proband underwent a testicular biopsy revealing rare spermatogonia and Leydig cell hyperplasia. Despite steadily progressive doses of hCG and Pergonal administered over a 3-yr period, the proband remained azoospermic. The proband's mother, sister (obligate carrier), and niece all had a history of delayed puberty, with menarche occurring at ages 17-18 yr. Baseline patterns of pulsatile gonadotropin secretion and gonadotropin responsiveness to exogenous pulsatile GnRH were examined in the affected males. LH, FSH, and free alpha-subunit were determined during 12.5-24 h of frequent blood sampling (every 10 min). Both patients then received pulsatile GnRH (25 ng/kg) sc every 2 h for 6-7 days. Gonadotropin responses to a single GnRH pulse iv were monitored daily to assess the pituitary responsiveness to exogenous GnRH. In the proband, FSH and LH levels demonstrated a subtle, but significant, response to GnRH over the week of pulsatile GnRH therapy. Free alpha-subunit levels demonstrated an erratic pattern of secretion at baseline and no significant response to pulsatile GnRH. We conclude that 1) affected males with AHC/HH may have an intrinsic defect in spermatogenesis that is not responsive to gonadotropin therapy; 2) female carriers of DAX1 mutations may express the phenotype of delayed puberty; and 3) although affected individuals display minimal responses to pulsatile GnRH, as observed in other AHC kindreds, subtle differences in gonadotropin patterns may nevertheless exist between affected individuals within a kindred.

Suppression of the growth hormone (GH) response to clonidine and GH-releasing hormone by exogenous GH.

GH release in response to clonidine and human GH-releasing hormone-(1-44) (hGHRH-44) was assessed in 11 boys (aged 7-14 yr) with short stature, who had normal GH secretion. The response to these 2 provocative stimuli was repeated after, respectively, 2 and 3 days of treatment with human GH (0.1 U/kg, im). Exogenous GH significantly blunted the response to both clonidine [the mean 2-h integrated serum GH concentration falling from 1050 +/- 350 (+/- SEM) to 749 +/- 297 ng/ml X min; P = 0.03] and hGHRH-44, the 2-h integrated GH concentration falling from 1553 +/- 358 to 547 +/- 202 ng/ml X min; (P = 0.03). Plasma insulin-like growth factor (IGF-II) concentrations did not change after GH administration. In contrast, plasma IGF-I (somatomedin-C) concentrations increased from 97 +/- 16 ng/ml before administration of GH to 142 +/- 32 ng/ml (P = 0.05) after two days and 149 +/- 23 ng/ml (P less than 0.01) after the third treatment day. However, no correlation was found between the changes in response to clonidine or hGHRH-44 and changes in circulating levels of IGF-I. Our data confirm the existence of GH-dependent feedback inhibition of GH release during childhood and suggest that this inhibition operates, at least in part, at the level of the pituitary. While participation of the IGFs/somatomedins in this feedback loop cannot be excluded, the inhibitory effects of exogenous GH do not depend directly on circulating plasma IGF-I or IGF-II levels.

Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia 2A.

The carrier status of 39 at-risk individuals in 6 multiple endocrine neoplasia 2A families was determined using a DNA based test. We were able to calculate a virtual diagnosis (probability greater than 95%) for 77% of the individuals and a probable diagnosis (probability greater than 90%) for 90% of the individuals. This study points out some of the problems of specific pedigree structures that can affect the risk calculation. This study further shows that no single test based on either biochemistry, pathology, or genetics can consistently and unambiguously produce a presymptomatic diagnosis. We also describe two specific examples where DNA testing has helped to resolve clinical uncertainties in at-risk individuals.

Hormonal studies in obligate heterozygotes and siblings of patients with 11 beta-hydroxylase deficiency congenital adrenal hyperplasia.

Hormonal response to ACTH stimulation and HLA genotyping were determined in families of patients with 11 beta-hydroxylase deficiency congenital adrenal hyperplasia. Neither hormonal measurements nor HLA genotyping were useful for the detection of heterozygosity in the families.

Long-term therapy of viramin D-resistant richets with 25-hydroxycholecalciferol.

The long-term effects of the vitamin D metabolite, 25-hydroxycholecalciferol (25-HCC), were evaluated in 2 children with hypophosphatemic vitamin D-resistant rickets. Serial total balance studies demonstrated an apparent lack of correlation between the effects of the vitamin on intestinal absorption of calcium and phosphorus and both the onset of healing in 1 of the 2 patients treated with 5,000 to 7,500 u of the metabolite and the absence of demonstrable radiologic improvement in another patient in whom the final dosage was 20,000 u. per day. At first, the metabolite induced a positive calcium balance in both patients resulting largely from a reduction in intestinal calcium excretion. Despite a continued positive calcium balance, 1 of the 2 patients did not demonstrate further healing, while in the other patient healing was noted even when total calcium balance was negative. Serum phosphate levels did not return to normal in either patient, nor was phosphate excretion altered by 25-HCC. Serum alkaline phosphatase remained elevated in both. Serum immunoassayable parathyroid hormone levels were consistently normal to high-normal in the 2 patients throughout more than 24 months of observation. No instances of hypercalcemia and only occasional hypercalciuric episodes were noted.

Outpatient treatment of juvenile-onset diabetes with a preprogrammed portable subcutaneous insulin infusion system.

Seven patients with juvenile-onset, insulin-dependent diabetes (aged 13 to 32 years) were continuously treated for 12 to 32 weeks while out of the hospital in their usual environment with a portable, battery-powered infusion pump which delivers insulin subcutaneously in basal (between-meal) doses with pulse dose increments before meals. Mean blood glucose levels (237 +/- 28 mg/dl during conventional insulin therapy) fell to 105 +/- 5 mg/dl after four weeks of pump treatment (p less than 0.01) and were maintained at 80 to 104 mg/dl as pump treatment was continued beyond eight weeks. Glycosylated hemoglobin levels (16.0 +/- 1.5 per cent before pump therapy) also fell within two weeks (p less than 0.01) reaching normal values (9.9 +/- 0.3) after eight weeks of pump therapy. Mean plasma cholesterol and triglyceride levels were elevated during conventional therapy and fell to normal after pump treatment. After the first month of pump treatment, only minor adjustments in insulin dose (less than 5 per cent of total daily dose) were made. No episode of mechanical pump failure occurred during the 1,110 patient-days of treatment. Overinsulinization and underinsulinization due to human error were relatively rare (four and six episodes, respectively) and failed to result in symptoms of hypo- or hyperglycemia. All patients performed their usual home, work or school activities during pump treatment. We conclude that normalization or near normalization of blood glucose levels can be achieved with a portable subcutaneous insulin infusion system when continuously used to treat patients with juvenile-onset, insulin-dependent diabetes outside the hospital for three to eight months.

Clinical research: assessing the future in a changing environment; summary report of conference sponsored by the American Medical Association Council on Scientific Affairs, Washington, DC, March 1996.

Concerns about funding of clinical research underlie all other problems identified at the Council on Scientific Affairs conference. Future National Institutes of Health (NIH) budgets are likely to be constant at best, and the general public expects cost containment to be an ongoing goal; this is exacerbated by the impending Medicare Trust Fund crisis. Meanwhile, traditional financial support of clinical research in academic medical centers (AMCs) through cross-subsidization is imperiled by competitive pressures largely caused by managed care. Although managed care organizations (MCOs) are potentially rich sources of funding and other resources, and some not-for-profit companies are conducting some research, for-profit MCOs have not demonstrated an understanding of the importance of clinical research. Young physicians are being discouraged from careers as clinical researchers and established investigators are "dropping out" because of demands for clinical productivity and competition for research grants, loss of patients/research subjects to managed care, perceived lack of status and compensation, and overall uncertainty about continued financial support. Efforts to assist current and potential clinical investigators are discussed in this report. Loss of patients, denial of reimbursement, and competition with MCOs and contract research organizations (CROs) have placed AMCs under unprecedented pressure. However, research centers located in AMCs have allowed investigators to conduct clinical research by providing a "protected environment." Furthermore, many AMCs are determined to continue conducting clinical research and are addressing related problems. Although the NIH will continue to be a major source of funding for clinical research, partnerships between various private and public entities provide important opportunities to maximize the productivity of all individuals and institutions involved. Potential partnerships include MCOs, AMCs, CROs, pharmaceutical companies and other industry, the Department of Defense, the Veterans Health Administration, practice-based physicians, and private foundations and patient support groups. "Partnerships in advocacy" for clinical research will be essential. Efforts to recruit for-profit MCOs to the clinical research endeavor identified in this report include (1) emphasizing issues of interest to them (eg, outcomes research); (2) stressing the significance of some research to the marketplace; (3) developing criteria to distinguish individual MCOs on the basis of their contribution to the public interest; (4) equating money spent on research with "R&D dollars" spent in nonmedical business enterprises; and (5) educating purchasers of health care (eg, corporate health plan directors) about clinical research. Conducting clinical research in all managed care settings requires leadership, the understanding and cooperation of physicians and support staff, wise use of limited resources (ie, funding only the best research projects), sound methodology, and above all, the perception that the research will ultimately improve patient care.

Psychosocial adjustment of latency-aged diabetics: determinants and relationship to control.

The relationship of psychosocial adjustment, family functioning, self-esteem, and diabetic control was studied in 20 latency-aged diabetic children and their parents. Moderate to severe adjustment problems were found in 11 (55%) of the patients. Child self-esteem, parental self-esteem, and family functioning, as scored by standard instruments, were all significantly greater in the group of children considered to be well-adjusted as compared to the maladjusted group (P less than .05 to .001). Of these, parental self-esteem appeared to correlate most closely with the child's adjustment. Twenty-four-hour urinary glucose excretion was two- to threefold greater in maladjusted as compared to well-adjusted patients (71 +/- 20 vs 20 +/- 5 gm, P less than .05). These data suggest that psychosocial adjustment problems frequently occur in latency-aged children with diabetes, are associated with poorer chemical control, and require a family-centered approach to intervention and management.

Presence of H-Y antigen in patients with Ullrich-Turner syndrome and X-chromosome rearrangements.

Cells from eight of ten patients with gonadal dysgenesis and an isochromosome for the long arm of X, (i(Xq)), have been found to be H-Y antigen-positive, using an assay that employs rat antiserum and Raji cells. In addition, two patients with del(Xq) were also found to be H-Y antigen-positive, whereas four patients in whom only a 45,X line was detected were H-Y antigen-negative. These findings suggest that the X chromosome plays a role in the expression of H-Y antigen in the absence of a Y chromosome. Since our patients with i(Xq) show no evidence of testicular differentiation, it is clear that there is not enough H-Y antigen on these patients' cells to direct the development of a testis. These findings are consistent with the view that the normal functioning of genes on the X and the Y chromosomes is necessary for testicular organogenesis to occur.

Oocyte production in a Turner syndrome patient with serologically detectable male antigen.

A patient with a 45,X/46,X, ring (X) karyotype has been found with serologically detectable male antigen, functioning ovaries, and documented oocyte production.