The impact of antibodies on clinical outcomes in diseases treated with therapeutic protein: lessons learned from infantile Pompe disease.

PURPOSE: Enzyme replacement therapy with rhGAA (Myozyme®) has lead to improved survival, which is largely attributable to improvements in cardiomyopathy and skeletal muscle function. However, crossreactive immunologic material-negative patients have a poor clinical response to enzyme replacement therapy secondary to high sustained antibody titers. Furthermore, although the majority of crossreactive immunologic material-positive patients tolerize or experience a downtrend in anti-rhGAA antibody titers, antibody response is variable with some crossreactive immunologic material-positive infants also mounting high sustained antibody titers. METHODS: We retrospectively analyzed 34 infants with Pompe disease: 11 crossreactive immunologic material-negative patients, nine high-titer crossreactive immunologic material-positive patients, and 14 low-titer crossreactive immunologic material-positive patients. Clinical outcome measures were overall survival, ventilator-free survival, left ventricular mass index, Alberta Infant Motor Scale score, and urine Glc(4) levels. RESULTS: Clinical outcomes in the high-titer crossreactive immunologic material-positive group were poor across all areas evaluated relative to the low-titer crossreactive immunologic material-positive group. For the crossreactive immunologic material-negative and high-titer crossreactive immunologic material-positive groups, no statistically significant differences were observed for any outcome measures, and both patient groups did poorly. CONCLUSIONS: Our data indicate that, irrespective of crossreactive immunologic material status, patients with infantile Pompe disease with high sustained antibody titer have an attenuated therapeutic response to enzyme replacement therapy. With the advent of immunomodulation therapies, identification of patients at risk for developing high sustained antibody titer is critical.

Detection of mono- and di-hexoses as metabolites of 4-bromoaniline using HPLC-TOF-MS/MS.

1. The metabolic fate of 4-bromoaniline (4-BrA) was investigated in rat following intraperitoneal administration at 50 mg kg(-1) using HPLC-TOF-MS/MS. 2. The sensitivity provided by the use of TOF-MS/MS, aided by the distinctive isotope pattern resulting from the presence of the bromine substituent in the molecule, enabled the detection of many previously uncharacterized metabolites in the samples. 3. Several groups of minor metabolites were detected in the urine that corresponded to a number of isomeric hexose and di-hexose-containing conjugates (possibly glucosides and diglucosides) of 4-BrA. 4. As well as hexose and di-hexose conjugates of 4-BrA, several further groups of metabolites that also contained either a sulphamate or sulphate group in addition to the sugar moieties were also detected.

Disposition of D-[U-14C]tagatose in the rat.

The purpose of this experiment was to determine the disposition of D-tagatose, under development as a low-calorie sweetener, in conventional and germ-free male rats. One group of conventional rats was fed a diet containing D-tagatose (100 g/kg) mixed with the nonpurified diet (900 g/kg) for 28 days. Then, [U-14C]-labeled D-tagatose was administered as a single dose (approximately 220-380 kBq) to 4 of these adapted rats, as well as to 15 conventional and germ-free rats with no prior exposure (i.e., unadapted) to D-tagatose. Eleven of the 19 dosed animals (4 adapted conventional, 3 unadapted conventional and 2 unadapted germ-free, all dosed orally, plus 2 unadapted conventional dosed intravenously) were placed in metabolism chambers and samples of CO2, urine, and feces taken at regular intervals. At termination, a complete material balance was obtained based on the recovery of 14C. Over the 6-h digestive period, D-tagatose was metabolized to release 39.9 and 13.9% of the oral dose as CO2 in the adapted conventional rats and in the unadapted germ-free rats, respectively. Total releases approximated 68 and 22%, respectively. The difference in CO2 evolution is ascribed to microbial fermentation of D-tagatose in the gut of the conventional rats. The role of adaptation was confirmed by finding 93% less D-tagatose in the feces of the adapted conventional rat than in the feces of the unadapted conventional rat. The intestinal absorption of D-tagatose in the rat is estimated to be 20%. The results demonstrate that D-tagatose is metabolized primarily by microorganisms in the gut of the rat, with an upper limit between 15 and 20% of oral dose metabolized by the host.

[Level of certain carbohydrate components in blood serum and urine of patients stomach and duodenal ulcer patients in the late post-vagotomy period]. / Soderzhanie nekotorykh uglevodnykh komponentov v syvorotke krovi i moche bol'nykh v otdalennye sroki posle vagotomii pri iazvennoi bolezni zheludka i dvenadtsatiperstnoi kishki.

Estimation of protein-bound hexoses, fucose and sialic acids in the blood and urine of 64 patients with gastroduodenal ulcers within later periods after various types of gastric vagotomy showed that the denervation inhibited the protective barrier of gastric and duodenal mucosal membrane. Protective functions of gastroduodenal mucosal membrane were most distinctly inhibited after selective proximal vagotomy which may be responsible for higher amounts of recurrences of ulcerous disease after this type of vagotomy than other types of denervation.

Increased urinary excretion of acid mucopolysaccharides and glycopeptides in hypothyroidism following thyroid hormone therapy.

Urinary excretion of acid mucopolysaccharides and glycopeptides in the urine of an untreated patient with cretinism were measured before and after thyroid hormone replacement. Urinary uronic acid and hexose excretion in the CPC-precipitated fraction increased four to ten times after thyroid hormone administration. The maximum excretion was observed after 2 months of thyroid replacement. The excreted acid mucopolysaccharides consisted of chondroitin sulphate A/C and minor quantities of heparan sulphate. Urinary excretion of glycopeptides, particularly small molecular glycopeptides rose also four to five-fold with thyroid hormone administration. These data suggest that thyroid hormone markedly influences the metabolism of acid mucopolysaccharides and glycoproteins. Possibly, the lack of thyroid hormone caused a decreased activity of various lysosomal glycosidases and sulphatases.

Measurement of hydroxylysine glycosides in urine and its application to spinal cord injury.

A new technique to evaluate the degradation of skin or bone collagen by measuring glucosylgalactosyl hydroxylysine and galactosyl hydroxylysine is presented. The method utilizes an automated amino acid analyzer. Eluents used are lithium buffers, and the color reagent is ninhydrin. Both glycosides elute in 3.5 h. Samples require minimum preparation. Urinary concentrations of both glycosides in ten patients with cervical spinal cord injuries of less than six months duration were higher than in five healthy controls. Proportional increases were different for each of the two glycosides. Variations in the proportional increase of each glycoside indicate different rates of degradation of skin and bone collagen. Repeated evaluations of the two urinary glycosides may help to predict whether patients are likely to develop skin- or bone-related clinical complications.

Urinary glycoconjugates in schizophrenic patients.

The level of glycoconjugates excreted in the urine of five schizophrenic patients was compared to that excreted by six controls. Urinary samples were fractionated by means of gel filtration and anion exchange chromatography to yield (i) fraction I consisting of basic, neutral, or slightly acidic glycopeptides and/or oligosaccharides, (ii) fraction II consisting of acidic glycopeptides and/or oligosaccharides, and (iii) fraction III consisting of glycosaminoglycans (acidic mucopolysaccharides). The hexose levels of fraction II (p less than 0.05) and uronate levels of fraction III (p less than 0.025) were significantly reduced in schizophrenic patients. The ratio of galactose/mannose in the glycoconjugates of fraction II was lower than normal in the urine from schizophrenic patients. Significantly (p less than 0.05) lower levels of rhamnose and higher levels of fucose were found in the glycoconjugates of fraction I from schizophrenic patients. Contrary to a previous report, we found no evidence for the presence of an abnormally elevated rhamnose-containing glycoprotein or glycoconjugate in fraction II. I appears that the pattern of metabolism of glycoproteins and glycosaminoglycans in schizophrenic patients deviates from the normal.

Glucose-containing oligosaccharides in normal human urine.

Normal human urine was treated with cetylpyridinium chloride (CPC), and the CPC-supernatant was passed through columns of Dowex 50 X 2 (H+ form) and Dowex 1 X 2(Cl-form) in succession. The concentrated effluent containing neutral saccharides was then gel-filtered through a column of Sephadex G-10. The resulting effluent was divided into 5 subfractions (SFs. 1-5). Comparison of hexose contents of the subfractions indicated that healthy individuals regularly excreted neutral oligosaccharides in urine with a similar pattern regardless of age or sex. Component A isolated from SF2 was characterized as glucosylgalactose by paper chromatography before and after acid hydrolysis of the intact one and its reduction product.

Sialic acid-containing glycoconjugates in human lactation urine.

The excretion rate of neutral hexoses, fucose and sialic acid of oligosaccharide and glycopeptide fraction has been determined in lactation urine. Thin layer chromatography has been used to investigate the excretion pattern of oligosaccharides, specially sialyl-oligosaccharides, during the course of lactation and after weaning. Lactation urine contains sialyloligosaccharides which are not present in normal urine and their presence has been studied up to the 3rd week after weaning. These results indicate a mammary origin for most of these compounds and confirm the hypothesis of previous research.

Urinary glycopeptides and glycosaminoglycans in children and adults as affected by age.

The glycopeptide and glycosaminoglycan excretion was studied in the urine of normal human subjects from newborns to 68 years of age. The glycopeptide and glycosaminoglycan preparations were made by digesting the urine with peoteolytic enzymes followed by removal of enzyme and undigested protein and finally freeze-drying of the supernatant. The contents of hexose, sialic acid and uronic acid were determined on these preparations. Because of the striking differences in body weight all values were expressed in terms of milligrams per 24 hours per kilogram of body weight. When corrected for body weight, all of the carbohydrate levels were significantly higher in infants and young children under 5 years of age than in older children and adults. No other significant changes with age were found after the age of 7 years. There were no significant differences between the sexes at any age.

Further studies on gel filtration of urinary substances.

Urine specimens from healthy children were concentrated and filtered through Sephadex G-100 and G-25. The eluate was recovered in fractions of 5 ml each. Total hexoses, galactose, total proteins and total uronic acids were determined on each fraction. Total hexoses and galactose were eluted in 6 distinct peaks. The uronic acid containing GAG were eluted in 4 small peaks on G-25 and 1 peak on G-100. This peak is eluted always with high amounts of galactose containing glycoproteins.