Hy Goldman

Use of Phosphate and Vitamin D to Prevent Dwarfism and Rickets in X-Linked Hypophosphatemia

Abstract Eight children with X-linked hypophosphatemia (three girls and five boys between three and 15 years of age) were treated for a total of 11,297 patient days with an inorganic phosphate salt...

An inherited disorder of isoleucine catabolism causing accumulation of alpha-methylacetoacetate and alpha-methyl-beta -hydroxybutyrate, and intermittent metabolic acidosis.

Extract: At least 15 apparently inherited disorders of branched chain amino acid catabolism are now known; the 12th in chronological order of discovery is described in this report. It is a partial defect of the pathway of isoleucine oxidation beyond the level of oxidative decarboxylation and prior to the oxidation of propionate. The impairment of isoleucine catabolism appears to be situated at the “thiolase” reaction which converts α-methylacetoacetyl coenzyme A (CoA) to propionyl-CoA and acetyl-CoA.Two pedigrees (B and M) were investigated in detail. A third (S pedigree) has been brought to our attention for analysis of metabolites in urine but we have not performed additional studies in the latter. Each propositus was ascertained because of intermittent, odorless metabolic acidosis usually precipitated by intercurrent infection. Lethargy and coma occurred frequently during the periods of acidosis. One M sib, also presumably affected, died abroad in such an episode. Symptoms can be ameliorated by a low protein diet and careful attention to the management of intercurrent illness.A large excess of α-methyl-β-hydroxybutyrate and a seemingly smaller excess of α-methylacetoacetate is present at all times in the urine of the three propositions. The M and S propositi also excrete N-tiglylglycine. The amounts of these unusual metabolites increase severalfold during acidosis and after a dietary load of l-isoleucine (75 mg/kg, 3 times daily for 2 days). The urine also contains butanone, particularly during acidosis. The amount of propionate and of glycine and other amino acids in blood and urine is always normal in our patients. Oxidation of l-isoleucine-U-14C to CO2 by cultured skin fibroblasts is about 45% of normal in the B propositus. The precise nature and location of the enzyme defect awaits clarification.Studies of family members reveal that presumed obligate heterozygotes excrete a small excess of α-methyl-β-hydroxybutyrate at all times; the amount can be increased by L-isoleucine feeding. The condition is apparently inherited in autosomal recessive fashion. It is likely that more than one form of mutant allele is responsible for the condition, as it is found in the three different pedigrees described here.Speculation: Investigation of “unexplained,” intermittent metabolic acidosis in childhood has led to the discovery of a “new” disorder of branched chain amino acid catabolism. Gas chromatography coupled with mass spectrometry were important aids to the diagnosis. Rapid escalation of acidosis during catabolic episodes encourages one to suspect that specific metabolites, themselves accumulating during episodic illness, may further inhibit the mutant enzyme. A temperature-sensitive mutant enzyme was not identified in cultured skin fibroblasts.

USE OF DITHIOTHREITOL TO CORRECT CYSTINE STORAGE IN CULTURED CYSTINOTIC FIBROBLASTS

Abstract Dithiothreitol (D.T.T.) removes cystine from cystinotic fibroblasts. The reagent is not toxic to cells at low concentrations (0.1 m M ). A cystinotic patient tolerated D.T.T. intravenously for 10 days with apparent reduction of cystine storage in rectal mucosa. He died of uraemia 1 month later.

A Study of the Structural and Biochemical Development of Human Fetal Islets of Langerhans

The structural and biochemical ontogeny of 14 mid-trimester (12.0–19.5 wk) human fetal endocrine pancreata were examined. Each pancreas was halved at the region of the isthmus into duodenal (D) and splenic (S) portions, and islets, isolated from each of the two portions, were aliquoted and grouped according to diameter into 4 categories: (1) > 300 μm (2) 300–200 μm (3) 200–100 μm (4) 100 μm. At all fetal ages, the number of islets isolated from the splenic half was greater than from the duodenal half, and the number of islets increased as their diameter decreased. The mean total cell number per islet for each of the 4 categories are reported: (1)(D) 30,810 (S) 28,450; (2)(D) 10,970 (S) 10,250; (3)(D) 3,390 (S) 3,460; (4)(D) 1,930 (S) 1,840. Measurement of islet insulin (IRI), glucagon (IRG), and somatostatin (IRS) by radioimmunoassay showed a progressive increase with time in the content of all three hormones in all islets from both halves of the pancreas (with the exception of the smallest islets of <100 μm diameter). After 18 wk, a dramatic increase in the content of all three hormones was observed particularly in the case of the largest (>300 μm) islets. In contrast, pancreatic polypeptide (IRPP) shows no significant increase. With the exception of the smallest islets (100 μm), all splenic-half islets contain a significantly greater content of IRI, IRG, and IRS compared with their duodenal-half match while the content of IRPP is significantly greater in the duodenal-half islets. In addition, all islets with the exception of the largest (>300 μm), contained equal quantities of IRG and IRS and these results were apparent at 12 wk gestation. These data provide new insights into the ontogeny of individual islet cells during the mid-trimesteric period and serve as a basis for future studies of both normal and abnormal pancreatic development.

The in vivo use of dithiothreitol in cystinosis.

Summary: Two male patients with late stage (uremic) infantile nephropathic cystinosis (INC) (Table 1) were treated by mouth with the reducing agent dithiothreitol (DTT), at doses not exceeding 25 mg-kg-1 body weight three times per day. Three sequential periods of observation were obtained in both patients: on thiol (8.5 months); off thiol (8–9 months); on thiol again (7 months or longer). Other than nausea and vomiting at the maximum dose range, no apparent toxicity was observed. One subject died in uremia in the 24th month of the study.The half-cystine concentration in peripheral blood leukocytes decreased during both treatment periods in each patient from initial pretreatment levels in excess of 8 nmol·mg-1 protein (normal <0.1 nmol mg-1) to 10–20% of initial values (Table 2 and Fig. 1, A and B). Reduction in total number of blood leukocytes or in the neutrophil fraction, where cystine storage occurs selectively in cystinosis, did not occur (Table 3) as a possible explanation for these findings; nor did storage of samples, a possible artifact, influence the cystine content of cystinotic cells (Fig. 2).Multiple site rectal mucosa biopsy clearly revealed cystine storage but serial biopsies did not reflect a positive DTT response when compared with the leukocyte assay (Table 4). High intersample variation in cystine content, even between samples taken at one time, prevented measurement of a treatment response.DTT had no apparent detrimental effect on the concentration of representative proteins, including hemoglobin (Table 3), serum insulin, and serum immunoglobulin during the treatment trials. Renal function (glomerular and tubular) was severely depressed and did not improve during the period of observation in either patient (Table 2; Fig. 3, A and B).Postmortem tissues from one patient revealed 10–40-fold excess cystine accumulation in kidney cortex and liver (Table 5). However, these levels of accumulation are at the lower range of or even below published values for cystine in cystinotic kidney and liver.Whereas chemical methods are not reliable for detecting and measuring DTT in biologic fluids, preliminary evidence indicates that a silylated derivative of oxidized DTT can be detected in the urine of patients receiving DTT by mouth (Fig. 4). This finding suggests that the thiol is absorbed and excreted.Speculation: The defect in cystine metabolism (or transport) in cystinosis remains unknown. However, if administered early in the course of INC, DTT might prevent the occurrence of irreversible phenotypic components by preventing cystine accumulation.

Dominantly inherited osteogenesis imperfecta in man: an examination of collagen biosynthesis.

Extract: We have examined control subjects and patients in an effort to discover a metabolic basis for dominantly inherited osteogenesis imperfecta (OI). Studies were carried out in vitro with cultured skin fibroblasts obtained from OI patients, and in vivo on peptide-bound hydroxyproline excretion in urine. Urinary hydroxyproline excretion (milligrams/24 hr) adjusted for age is essentially normal in OI patients, although the mean excretion rate is below average. The latter finding is presumably a reflection of the smaller body mass of OI patients.The OI skin fibroblasts, matched for age of donor, site of biopsy, phase of growth, and generation number in culture, incorporate L-proline into hot trichloroacetic acid (TCA)-soluble protein (collagen) at normal rates. The rate of conversion of proline to hydroxyproline in the nascent polypeptide is also normal in OI. Incorporation of L-lysine was also normal in OI. These findings indicate that peptide synthesis of collagen is not impaired in OI.Rates of galactose incorporation into collagen and the extractability of collagen into normal saline or 0.2 M citric acid were all normal both in OI cells and in the culture medium recovered from the monolayer. These findings, in combination with the urinary data on hydroxyproline excretion in vivo reveal that cross-linking and export of collagen in OI is essentially normal.The elution profile after ion exchange chromatography of fibroblast collagen on carboxymethyl (CM)-Sephadex was also examined. The normal 2/1 ratio of peak 1 (largely α1(I) chains) to peak 2 (largely α2 chains) was found in OI fibroblast extracts, which implies that synthesis and initial aggregation of the two types of polypeptide to yield [α1(I)]2α2 collagen composition is not abnormal in OI.Despite the negative biochemical findings, a consistent defect in the morphology of OI cells was identified in the log phase and the confluent phase of monolayer cultures. The finding is characterized by irregular packing of the aggregated cells and by an irregular tessellated appearance of the individual OI fibroblast. This observation reassures us that the inherited defect is expressed in vitro.Speculation: An abnormality in the primary sequence of polypeptide chain in collagen would be compatible with all of our findings and with the genetics of OI. The mutant allele would affect only about half the products, under the control of only one of the loci determining the polypeptide sequences in collagen chains. Because the OI allele is not expressed in cartilage, a tissue without α2 collagen chains, the defect in OI would perhaps be found in the α2 polypeptide. However, since the α1(II) chain of cartilage differs in amino acid composition and in hydroxylysine-linked carbohydrate from the α1(I) chains of noncartilagenous structures, a defect in α1(I) chains at the nonhomologous residues will also require investigation.

Spontaneous diabetes mellitus syndrome in the rat. III : pancreatic alterations in aglycosuric and untreated diabetic BB Wistar-derived rats

The pancreatic alterations in aglycosuric and untreated diabetic BB Wistar-derived rats are described. A common finding, often seen in young aglycosuric rats, is that of discrete foci of periductular and/or acinar aggregates of lymphocytes and macrophages. Sites of periductular mononuclear cell infiltrates usually lack endocrine cells. In contrast, foci of acinar infiltrates, although distinct from the predominant endocrine cell mass in the islets of Langerhans, often contain small numbers of alpha and/or beta cells. It is suggested that these clusters of endocrine cells may in some way be antigenically different from those resident in the principal islets and thus serve as an additional target for the immune system in rats bearing the BB genome. The development of overt diabetes requires a massive destruction of beta cells within the islets of Langerhans. Two forms of diabetes mellitus emerge in untreated animals. The more common, designated unstable diabetes, is severe and lethal unless treated with insulin. Less commonly, a stable type of diabetes mellitus ensues for which insulin therapy is not mandatory. In each, the concentration of pancreatic immunoreactive insulin is profoundly decreased, although relatively greater amounts are present in the stable form. Unstable diabetic rats demonstrate a reduction in the concentration of pancreatic immunoreactive glucagon and somatostatin, suggesting that alpha and delta cells also sustain injury in this model of insulin-dependent diabetes mellitus.

Exchange transfusion in the treatment of hepatic coma

Death due to acute hepatic failure is reported in 2 siblings despite therapy with a total of 38 exchange transfusions. Extensive laboratory documentation is presented and new data discussed. This experience and a comprehensive review of exchange transfusion in hepatic coma provide the basis for a critical assessment, of this mode of therapy.

Replication of cytomegalovirus in human thymic epithelial cells

Cytomegalovirus (CMV) has often been cited as a cause of immune suppression in children, yet little is known of the mechanisms through which this agent might affect immune function. We have succeeded in using CMV to productively infect cultured human fetal and infantile thymic epithelial (TE) cells. Morphological changes were apparent by 2–4 days after viral inoculation. CMV-related early antigen (EA) and late antigen (LA) were detected by immunofluorescence after 8 days, and progeny infectious CMV was recovered from culture media after 12–17 days. TE cells that reacted with monoclonal antibodies specific for keratin and for GQ ganglioside were predominant throughout the culture period. In contrast, infection by CMV resulted in a significant decrease in numbers of cells reactive with monoclonal antibodies specific for mesoderm-derived components. Inoculation of TE cells with CMV also caused a diminution in levels of detectable interleukin-1 (IL-1)-related antigen by 17 days after infection.

X-linked hypophosphatemic rickets: A PTH-insensitive transport effect responsive to phosphate

Six children (2F, 4M) have been treated up to 6 years with a phosphate supplement by mouth 5 times daily (1–4g Pi/d). This regime required 27 home visits/pt/year but yielded a serum Pi conc. of 3.75 ± 0.8 mg% for the group. This in turn achieved: 1) complete healing of rickets in all; 2) satisfactory bone density; 3) marked catch-up growth in 5 of 6 patients. Serum PTH was normal in 4 previously untreated patients (mean = 35 μl Eq/ml, normal <40), but was raised during phosphate treatment (mean = 150 μl Eq/ml; 6 patients, 11 determinations). Secondary hyperparathyroidism with bone signs and hyperaminoaciduria occurred in 3 patients, reduction of phosphate and use of vitamin D2 (50–100,000 u/d) suppressed parathyroid hyperactivity. Net tubular reabsorpiton of phosphate studied intensively in one patient was constantly saturated at 39–50 μmoles/100 ml GFR (normal <90) over a serum Pi range from 1.5 to 7.3 mg%, and at endogenous serum PTH concs. varying between 28 and 490 μl Eq/ml. A primary renal disorder of phosphate reabsorption, which is insensitive to PTH and can be offset by dietary phosphate suplements, is proposed for this X-linked trait.