Laparoscopic removal of virilizing hilar cell tumor in a postmenopausal patient.

A postmenopausal woman experienced rapidly progressing hirsutism and signs of virilization. Hormone evaluations showed markedly elevated serum testosterone levels and no evidence of excess cortisol or dehydroepiandrosterone sulfate production. A computerized tomographic scan of the adrenals and ovaries was normal, and transvaginal ultrasound revealed a left ovary with a maximum diameter of 3.2 cm. At outpatient laparoscopic bilateral oophorectomy, the left ovary had a benign, 2.5-cm Leydig cell tumor, hilar cell variant. Laparoscopy may be useful in the diagnosis and treatment of select cases of virilizing tumors of the ovary.

Postoperative acute adrenal failure caused by transient corticotropin deficiency.

BACKGROUND: Acute adrenal insufficiency after a surgical procedure or trauma is rarely reported. In recent years, however, we have treated seven patients with acute primary adrenal insufficiency and three patients with secondary adrenal insufficiency who presented with shock after a surgical procedure or trauma. The standard cosyntropin test was misleading for the diagnosis of corticotropin deficiency. METHODS: In this study we measured serum cortisol in patients older than 65 years who had unexplained hypotension after an abdominal surgical procedure. If the serum cortisol was less than 15 micrograms/dl, we performed 1 microgram and standard (250 micrograms) cosyntropin tests and measured thyroxine, thyrotropin, leutinizing hormone in all patients, and free testosterone in men. RESULTS: We identified five (5%) of 105 patients after an operation who displayed evidence of corticotropin deficiency (i.e., serum cortisol < 15 micrograms/dl during hypotension, prompt hemodynamic improvement with glucocorticoid therapy, and normal response to standard dose cosyntropin). In these patients 1 microgram cosyntropin produced abnormal peak cortisol levels. These patients also had thyrotropin or leutinizing hormone deficiency. After recovery the low hormone levels improved or became normal. CONCLUSIONS: Postoperative adrenal insufficiency, particularly that caused by transient corticotropin deficiency, is more common in patients than currently recognized. The 1 microgram cosyntropin test may be more sensitive than the standard test for identifying secondary adrenal insufficiency.

Transient corticotropin deficiency in critical illness.

We describe three critically ill patients who displayed indirect evidence of transient corticotropin deficiency. All these patients were elderly, were poorly nourished, and had unexplained hypotension intraoperatively or immediately postoperatively. During the hypotensive episodes, they had inappropriately low plasma cortisol levels (10, 12, and 6 micrograms/dl) and responded dramatically to the administration of glucocorticoids. A normal response to infusion of synthetic corticotropin excluded primary adrenal insufficiency. Two patients tested had low thyroxine levels without increased thyrotropin concentrations and depressed levels of gonadotropins. In all three patients, the dose of glucocorticoids was successfully tapered and then discontinued. After recovery, serum thyroxine levels increased, gonadotropins reverted to normal concentrations, and the administration of metyrapone to two patients demonstrated normal hypothalamic-pituitary-adrenal function. Cortisol levels of less than 15 micrograms/dl in critically ill patients suggest the presence of adrenal insufficiency. The infusion of synthetic corticotropin may not exclude adrenal insufficiency attributable to corticotropin deficiency. If direct tests of corticotropin reserve are impractical, treatment with glucocorticoids is warranted.

Recurrence of 131I-induced thyroid storm after discontinuing glucocorticoid therapy.

A 19-year-old woman with Graves' disease developed thyroid storm 8 days after radioactive iodine therapy. The clinical manifestations of thyroid storm promptly improved after treatment with large doses of propylthiouracil, potassium iodide, propranolol hydrochloride, and dexamethasone. Four days after discontinuing dexamethasone, the syndrome recurred and was corrected by reinstitution of the glucocorticoid. We conclude that dexamethasone was an important adjunct for treating thyroid storm and was effective mainly by reducing peripheral triiodothyronine production.

Biosynthesis of heparin. Enzymatic sulfation of pentasaccharides.

Heparin-derived pentasaccharides with the general structures GlcN-GlcA/IdoA-GlcN-GlcA/IdoA-GlcN (where GlcA represents D-glucuronic acid and IdoA represents L-iduronic acid) and GlcNSO3-GlcA/IdoA-GlcNSO3-GlcA/IdoA- GlcNSO3 (where -NSO3 represents an N-sulfate group) were tested as exogenous sulfate acceptors in incubations with adenosine 3'-phosphate 5'-[35S]phosphosulfate and microsomal enzymes from a heparin-producing mouse mastocytoma. No transfer occurred to the N-unsubstituted pentasaccharide containing only L-iduronic acid, but the other three isomers incorporated various amounts of 35S, which was totally present in N-sulfate groups. After complete chemical N-sulfation, all four pentasaccharides served as acceptors in O-sulfotransferase reactions and incorporated from 20 to greater than 200 times as much radioactivity as did the nonsulfated parent compounds. The C-6 position of the internal glucosamine unit was labeled preferentially, irrespective of the structures of the adjacent hexuronic acid units. Significant 2-O-35S-sulfation of IdoA units occurred in both -IdoA-Glc-NSO3-GlcA- and -GlcA-GlcNSO3-IdoA- sequences, whereas no significant sulfation of GlcA residues was detected. The pentasaccharide GlcNSO3-GlcA-Glc-NSO3-GlcA-GlcNSO3 thus can be used as a selective substrate in assays for glucosaminyl-6-O-sulfotransferase activity. The antithrombin-binding region, essential for the blood anticoagulant activity of heparin, has been identified as a pentasaccharide sequence with the predominant structure GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-++ +IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R represents either a sulfate or an acetyl group and -OSO3 represents an O-sulfate/ester sulfate group, with locations of O-sulfate groups indicated in parentheses) (Lindahl U., Thunberg, L., Bäckström, G., Riesenfeld, J., Nordling, K., and Björk, I. (1984) J. Biol. Chem. 259, 12368-12376). The products of [35S]sulfate transfer to the pentasaccharide GlcNSO3-GlcA-GlcNSO3-IdoA-GlcNSO3 contained molecules with high affinity for antithrombin, corresponding to 0.3-0.5% of the total label. Structural analysis suggested the occurrence of O-[35S]sulfate groups at both C-6 of the nonreducing terminal glucosamine unit and C-3 of the internal glucosamine unit. No products with high affinity for antithrombin were formed from the pentasaccharides that had a different monosaccharide sequence than the binding region; and moreover, these oligosaccharides appeared unable to incorporate glucosaminyl 3-O-sulfate groups. These findings point to the importance of the uronic acid sequence in the generation of the antithrombin-binding region of heparin.

Proteoglycans and hypertension: III: Aorta proteoglycans in Dahl salt-sensitive hypertensive rats.

The vascular proteoglycans probably have an important influence on the biomechanical properties of blood vessels and, therefore, may play a role in the development or maintenance of hypertension. In the aorta of the spontaneously hypertensive rat, the authors previously observed an increased content of chondroitin sulfate, an increased incorporation of [35S]sulfate into proteoglycans, and qualitative alterations in the [35S]polysaccharides compared to the normotensive Wistar Kyoto rat. To determine if these differences were related to hypertension or to strain variations, normotensive and hypertensive Dahl S rats were studied. There was a significant elevation (70%) in the aorta content of chondroitin sulfate, whereas the dermatan sulfate and hyaluronic acid contents were similar in the two groups. The in vitro incorporation of [35S]sulfate was increased 2.6-fold in the hypertensive animals. No differences between the two groups were observed with respect to the gel chromatographic profiles of the [35S]proteoglycans or the charge density of the [35S]glycosaminoglycans, as assessed by ion exchange chromatography. It was concluded that the increase in chondroitin sulfate and [35S]sulfate incorporation into proteoglycans occurred as a result of hypertension, regardless of genetic factors.

Proteoglycans and hypertension. II. [35S]sulfate incorporation into aorta proteoglycans of spontaneously hypertensive rats.

Spontaneously hypertensive (SH) rats are known to have an increased content of chondroitin sulfate (CS) proteoglycans (PG) in the aorta as compared to normotensive Wistar Kyoto (WKY) rats. In the present study we have compared WKY and SH rat aortas with respect to [35S]sulfate incorporation in vivo and in vitro. The specific activity (cpm/mg aorta) of the total glycosaminoglycan (GAG) pool from SH rat aorta, measured 48 h after intraperitoneal injection of [35S]sulfate, was twice as high as that of WKY aorta GAG. After in vitro incubation of aortas for 4 or 6 h, the specific activity (cpm/mg aorta) of glycosaminoglycans from SH rat was 2.4- to 7.1-fold higher than in controls. Labeled PG were extracted with 4 M guanidine from aortas which had been incubated with [35S]sulfate, and chromatography of the extract on Sepharose CL-6B yielded two incompletely resolved peaks, one emerging with the void volume (peak I) and one in a more retarded position (peak II). Peak I (WKY) contained nearly equal amounts of CS and HS (53 and 46%, respectively) and a small amount of DS (8%). Peak II (WKY) (Kav, 0.34) was divided into two fractions; the fraction of larger molecular weight (II A) contained 43% CS, 35% DS, and 20% HS, whereas the smaller fraction (II B) contained 40% CS, 51% DS, and 5% HS. In each corresponding pool from SH rat aorta, a similar proportion of HS was found, but the DS content was approximately half, and the CS content was correspondingly greater. The estimated molecular weights of the CS/DS chains in peaks I, II A, and II B from WKY aorta were 34,600, 18,800, and 11,600 daltons, respectively, whereas the corresponding values for the SH rat aorta pools were 32,300, 24,700, and 17,000 daltons, respectively. The proportions of 4- and 6-sulfated galactosamine residues as well as the degree of sulfation of the CS/DS PG were similar in the two strains. The HS-PG was larger in the WKY rat aorta and was made up of larger HS chains (Mr 26,600 vs. 16,100); however, the degree of sulfation was apparently similar in the two strains. These results suggest that the rates of PG synthesis and/or degradation and the PG structure are altered in the SH rat aorta.

Proteoglycans and hypertension. I. A biochemical and ultrastructural study of aorta glycosaminoglycans in spontaneously hypertensive rats.

The extracellular matrix of blood vessel walls contains elastin, collagen, and proteoglycans, all of which can affect vascular resistance and, hence, blood pressure by virtue of their biomechanical properties. In the present study, we have begun to explore the possibility that proteoglycans may play a role in the pathophysiology of hypertension by analyzing, qualitatively and quantitatively, the polysaccharide components of proteoglycans from aorta of two normotensive rat strains, Wistar Kyoto (WKY) and Wistar rats, and from spontaneously hypertensive (SH) rats of the Okamoto strain. The total concentration of aorta glycosaminoglycans in the SH rat was 33% higher than in the WKY rat, due to a 164% increase in chondroitin 4- and 6-sulfate. The content of dermatan sulfate (DS), hyaluronic acid (HA), and heparan sulfate (HS) was similar in the two strains. The 4-wk-old SH rat also had an increase in chondroitin sulfate (CS) compared to the 4-wk-old WKY rat, without any change in DS, HA, or HS. The Wistar rat had approximately the same concentration of CS und DS in the aorta as the WKY rat, but HS und HA were reduced by 62 and 37%, respectively. The galactosaminoglycans (CS and DS) were heterogeneous on cellulose acetate electrophoresis and exhibited a different pattern for each of the three strains. Undersulfated CS accounted for 15% of the total CS in WKY aorta but was present in only trace amounts in the SH aorta; 2% of the CS from the Wistar aorta was undersulfated. In all three strains, DS was exclusively 4-sulfated, and the CS contained approximately equal amounts of 4- and 6-sulfated galactosamine residues. Ultrastructural studies demonstrated that the HS was localized in the subendothelial matrix and the pericellular region surrounding the medial smooth muscle cells. CS and DS were primarily associated with collagen in the media. In the SH rat aorta the subendothelial matrix was thicker, and there was a relative increase in the CS/DS in the smooth muscle cell pericellular matrix. We suggest that, if similar alterations in CS proteoglycans are present in the resistance vessels, these changes may contribute to the increased peripheral vascular resistance in the hypertensive animal.

Oligosaccharide substrates for heparin sulfamidase.

The Sanfilippo A syndrome is characterized by a deficiency in heparin sulfamidase, which removes the N-sulfate groups of heparan sulfate and heparin in the course of normal catabolism of these polysaccharides. [N-35S]Heparin is the most commonly used substrate for the assay of sulfamidase activity but has certain disadvantages which have prompted us to search for alternative substrates. We report here on the use of heparin oligosaccharides for this purpose. The trisaccharide, GlcN-IdoUA-GlcN, and the pentasaccharide, GlcN-GlcUA-GlcN-GlcUA-GlcN, were N-sulfated with [35S]sulfur trioxide-trimethylamine complex; the tetrasaccharide, GlcN-UA-GlcN-UA, and the pentasaccharide, GlcN-IdoUA-GlcN-IdoUA-GlcN, were labeled by reduction with sodium borotritide followed by chemical N-sulfation. When incubated with sonicates of cultured skin fibroblasts from normal individuals, all four oligosaccharides were found to serve as substrates for heparin sulfamidase. Fibroblast sonicates from patients with the Sanfilippo A syndrome had little or no activity toward these substrates. Optimal activity of the enzyme was at pH 4.4-4.5. Comparison of the kinetic parameters showed that heparin had a lower Km than the oligosaccharides, whereas the Vmax values of the latter were higher than for heparin.

New oligosaccharides from heparin and heparan sulfate and their use as substrates for heparin-degrading enzymes.

Oligosaccharides were isolated from heparin and heparan sulfate by a procedure consisting of three major steps: (a) acid hydrolysis; (b) gel chromatography; and (c) cation exchange chromatography on an amino acid analyzer. To date, six new oligosaccharides have been isolated by this procedure and have been sequenced by a combination of NaB3H4-labeling and deaminative cleavage with nitrous acid. The structures of these oligosaccharides were as follows: 1. GlcN-GlcUA-GlcN 2. GlcN-IdUA-GlcN 3. GlcN-GlcUA-GlcN-GlcUA-GlcN 4. GlcN-IdUA-GlcN-GlcUA-GlcN 5. GlcN-GlcUA-GlcN-IdUA-GlcN 6. GlcN-IdUA-GlcN-IdUA-GlcN The linkage positions and anomeric configurations were assumed to be the same as in the polysaccharides from which the oligosaccharides originated. The usefulness of some of these oligosaccharides as enzyme substrates was tested after appropriate modifications and radioactive labeling. Oligosaccharides 2 and 3 were N-[35S]sulfated and were found to serve as substrates for heparan N-sulfate sulfatase (heparin sulfamidase), with a homogenate of cultured skin fibroblasts as enzyme source. Similarly, reduction of oligosaccharide 2 with NaB3H4 yielded a substrate for acetyl-CoA:alpha-D-glucosaminide N-acetyltransferase. Finally, the previously known disaccharide, 4-O-alpha-D-glucosaminyl-L-iduronic acid, which was isolated in the course of this work, was N-acetylated with [3H] acetic anhydride and was shown to be a substrate for N-acetyl-alpha-D-glucosaminidase.

Myoinositol metabolism in diabetes mellitus. Effect of insulin treatment.

The metabolism of myoinositol has been studied in 10 nondiabetic subjects and in six patients with diabetes mellitus before and after insulin therapy. While dietary myoinositol intake and fecal myoinositol excretion were similar in both groups, urinary myoinositol excretion was increased 10-fold in the untreated diabetic and accounted for a significant fraction of his dietary myoinositol intake. Insulin treatment restored the urinary myoinositol excretion toward normal. Despite increased myoinositol excretion, plasma myoinositol concentrations were significantly higher in the diabetics following the ingestion of a standard diet or of a 3.0-gm. myoinositol load. This abnormality in oral myoinositol tolerance was also corrected by insulin treatment. The size of the rapidly equilibrating myoinositol pool was significantly decreased in the untreated diabetic and returned to normal following a brief period of insulin treatment. The elevated plasma myoinositol concentrations observed following myoinositol ingestion in the uncontrolled diabetic presumably represents a combination of enhanced gastrointestinal absorption and impaired intracellular transport of myoinositol. The decreased space of distribution of myoinositol also suggests an impairment of intracellular myoinositol transport in the untreated diabetic. These observations are consistent with the speculation that hyperglycemia may condition a widespread relative intracellular myoinositol deficiency in man and suggest that restoration of normal intracellular myoinositol concentrations might prove to be of benefit in the prevention and treatment of certain of the complications associated with human diabetes mellitus.