Comparison of rates of hydrolysis of N-oleoyl and N-stearoyl glucocerebroside in patients with Gaucher's disease.

The deficiency of oleic acid as one of the fatty acids in glucocerebrosides that accumulate (31--77 mg/g dry weight) in the spleen in patients with Gaucher's disease was confirmed in 9 cases. In an effort to account for the 10-fold difference between the oleoyl glycocerebroside content of glucocerebrosides in spleen from controls and patients with Gaucher's disease, we compared the ability of extracts of spleen and fibroblasts from individuals with various forms of Gaucher's disease and controls to hydrolyze [14C]stearoyl and [3H]oleoyl glucocerebroside. The residual glucosylceramidase activity in patients with Gaucher's disease hydrolyzes the glucose moiety of oleoyl glucocerebroside at approximately the same rate as that of stearoyl glucocerebroside. Similarly, the more active glucosylceramidase of control tissue acts upon both oleoyl and stearoyl glucocerebrosides with equal efficiency. These observations indicate that a mutation affecting the substrate specificity of glucosylceramidase cannot account for the lack of oleic acid-containing glucocerebrosides in patients with Gaucher's disease. Thus, the hypothesis that the difference in fatty acid composition found in glucocerebroside is obtained as a result of a mutation affecting the specificity of the residual glucosylceramidase must be rejected.

Comparison of sodium and potassium intake with excretion.

Nine well-motivated adults, knowledgeable about nutrition, kept food records, saved food portions equal to what had been eaten, and collected 24-hour urine samples for 3 consecutive days. Estimates of sodium and potassium intake were calculated from food table analyses of written food records and from flame photometric analyses of food portions. For each subject the mean of the estimates for each of the 3 days was compared with the mean of urine analyses for sodium and potassium for each of the 3 days. For the group of nine subjects, the average estimate of sodium intake from analyses of food records was 11% lower than the average estimate of urinary sodium excretion; the average estimate of sodium intake from analysis of food portions was 2% higher than urinary sodium excretion. For individuals, there were large differences between estimates of intake and measurement of sodium excretion. For the group of nine subjects, the average estimate of potassium intake from analysis of food records was less than 1% lower than the average estimate of potassium urinary excretion; the average estimate of potassium intake from analysis of food portions was 13% higher than potassium urinary excretion. For individuals, as with sodium, there were large differences between estimates of intake and measurement of potassium excretion.

Metabolism of lysosomal enzymes in the protein-deficient weanling rat.

We have shown that the protein-deficient weanling rat fed a 3% casein diet, within 2 to 4 wk, exhibits marked changes in serum lysosomal hydrolases similar to those observed in children suffering from protein-calorie malnutrition: serum hexosaminidase, alpha-mannosidase, and beta-glucuronidase activities increase 3-fold, 2-fold, and 50%, respectively, whereas the acid phosphatase levels decrease by 50%. Rehabilitation of the protein-deficient animals with a diet containing 25% protein (i.e., casein) results in a rapid restoration of the plasma lysosomal hydrolase profiles to normal in less than 1 wk. The specific activities of various tissue lysosomal enzymes change significantly in the protein-deficient animals; however, no overall consistent pattern of change is apparent. In general, the greatest number of changes in lysosomal enzymes occurs in the kidney, whereas the brain exhibits the smallest differences between experimental and control animals in this regard. Perfusion experiments have shown that the rate of release of lysosomal enzymes from livers of rats fed the protein-deficient diet is profoundly altered when compared to that of control animals. Studies of the variation of enzyme secretion with time have demonstrated that the rate of secretion of hexosaminidase by the liver remains low and then rises markedly (3-fold) after the animals have been consuming the 3% casein diet for 16 days. In contrast, the secretion of both acid phosphatase and beta-glucuronidase is markedly depressed in the early phase of protein malnutrition (i.e., 7 to 16 days), and then increases greatly by the 3rd wk. These results demonstrate that changes occur in the rate of secretion of lysosomal enzymes by the liver during the course of experimental protein malnutrition.

Properties of cholesterol 7 alpha-hydroxylase in rat liver microsomal acetone powder.

Rat liver microsomes were extracted with acetone, and a microsomal powder preparation was obtained. The cholesterol 7 alpha-hydroxylase activity of acetone powder was linear with time, the amount of protein, and the amount of cholesterol in human or rat serum. Unesterified lipoprotein cholesterol was also an effective substrate, and the Km values increased progressively from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) to very-low-density lipoprotein (VLDL), suggesting that HDL-free-cholesterol was the better substrate.

CSF alpha 2-macroglobulin and C-reactive protein as aids to rapid diagnosis of acute bacterial meningitis.

alpha 2-Macroglobulin (AMG) and C-reactive protein (CRP) levels in cerebrospinal fluid (CSF) of patients with bacterial and aseptic meningitis have been analyzed by a rate nephelometric method to determine if these acute phase proteins can aid in differentiation of bacterial from aseptic meningitis. The mean CSF concentrations of AMG and CRP were 15 and 3.5 times greater, respectively, in the bacterial compared to the aseptic meningitis group. Also, the range of AMG levels showed minimal overlap between the two groups. The elevated levels of the proteins persisted after CSF cultures became negative. Quantitation of specific acute phase proteins in CSF may assist the differentiation of bacterial from aseptic meningitis.

An improved fluorometric leukocyte beta-glucosidase assay for Gaucher's disease.

Three fluorometric leukocyte beta -glucosidase assays were compared for their ability to diagnose Gaucher's disease and identify carriers of the disorder: the acid beta-glucosidase assay of Beutler and Kuhl [2], a pH 5.5-sodium taurocholate-dependent assay and a new procedure which employs conduritol B epoxide, an active-site specific inhibitor of glucocerebrosidase. All three assays unambiguously identified patients with Gaucher's disease. With regard to identifying carriers the bile salt dependent assay of Peters et al. and the conduritol B epoxide-dependent procedure gave the greatest discrimination between the mean beta-glucosidase values for the control and heterozygote samples when evaluated using Student's t test. The most reliable assay for the identification of the carrier state was the conduritol B epoxide-dependent procedure which can be expected to provide the fewest false negative results when classifying heterozygotes (5%). However, the fact that none of these methods will completely separate control and heterozygote samples indicates that their use in screening programs will result in a significant number of incorrect assignments.

Sensorineural hearing loss from quinolinic acid: a neurotoxin in middle ear effusions.

Quinolinic acid (QUIN) is an endogenous metabolite that exerts a neurotoxic effect by binding to specific neuronal receptors. Studies involving a broad spectrum of infectious and inflammatory central nervous system diseases have suggested a role for QUIN in causing neuronal injury. Since there is evidence for presence of the QUIN receptor in mammalian cochleas, QUIN was measured in middle ear effusions (MEEs). Gas chromatography/mass spectrometry detected QUIN in each of 65 diluted human MEEs, with a mean of 482 +/- 75 (SEM) nmol/L and a range from 15 to 2667 nmol/L. QUIN was also detected in each of 197 chinchilla MEEs from five different models of otitis media, with a mean of 10.6 +/- 1.3 (SEM) mumol/L and a range from 0.23 to 146.0 mumol/L (corrected for dilution). To determine whether QUIN causes sensorineural hearing loss (SNHL), QUIN solutions were placed on round window membranes (RWM) for 20 to 240 minutes, in 20 chinchillas. SNHL was detected by electrocochleography in QUIN-exposed animals, but not in saline controls. We conclude that QUIN is present in MEEs and that QUIN in the middle ear has the potential to cross the RWM and cause sensorineural hearing loss, possibly by binding to specific neuronal receptors in mammalian cochleas.

Hydrolase activity in middle ear effusions. Effect of antibiotic therapy.

Hydrolytic enzymes have been shown to be present in middle ear effusions recovered from children with both persistent and acute otitis media. In the present study, we investigated the effect of ampicillin therapy on the expression of hydrolytic enzyme activity in acute middle ear effusions using the chinchilla animal model. The median values of enzyme activities were lower for the ampicillin-treated animals when compared with the nontreated control animals. For the ampicillin-treated animals, eight of 12 assayed activities were characterized by a time-dependent decay of enzymatic activity. For the untreated animals, the majority of assayed activities (seven of 12) showed an increase in activity with time. These results show that sterilization of the middle ear cleft and elimination of the hydrolytic enzyme activity may be benefits of antimicrobial therapy and prerequisite to the healing of the inflamed mucosa.

Inhibition of lymphoproliferation by middle ear effusion in experimental otitis media.

In this study, experimental otitis media was created in the chinchilla by direct middle ear challenge with Escherichia coli endotoxin, Streptococcus pneumoniae, Haemophilus influenzae, or Pseudomonas aeruginosa. The effusions recovered from the chinchillas in all four challenge groups were shown to inhibit the lymphoproliferative response of chinchilla peripheral blood lymphocytes to stimulation with phytohemagglutinin. The effect was dose dependent, and for effusions of infectious origin, the degree of inhibition was directly related to the duration of infection. Presence of the inhibitor in plasma was undocumented, suggesting a local production within the middle ear. Lymphocytes from middle ears infected with bacteria but not middle ears challenged with endotoxin were hyporesponsive or nonresponsive to stimulation with phytohemagglutinin. These results confirm the presence of an inhibitor of the lymphoproliferative response in experimental otitis media of different etiologies.

Cytokines, immunoglobulins, and bacterial pathogens in middle ear effusions.

OBJECTIVE: To elucidate the role of cytokines, immunoglobulins, and bacterial pathogens in the middle ear effusions (MEEs) of children with otitis media (OM). DESIGN: Paired MEEs and serum samples collected from consecutive patients were assayed for immunoglobulins. Middle ear effusions were cultured for bacterial pathogens and assayed for interleukin-1 beta, interleukin-6, tumor necrosis factor alpha, and interferon gamma. The medical charts of the patients were retrospectively reviewed to define the history of OM. SUBJECTS: Seventy-five patients with a history of recurrent acute OM, persistent OM with effusion, or both. Exclusion criteria included the presence of a major coexisting condition, or an unclear or atypical history of OM. SETTING: A private practice at a tertiary care children's hospital. INTERVENTIONS: At the time of tympanostomy tube placement, with the patient under general anesthesia, one MEE and a serum sample were collected. RESULTS: Interleukin-1 beta was detected in 58% (44/75) MEEs; interleukin-6, 83% (60/72); tumor necrosis factor alpha, 37% (28/75) [corrected]; and interferon gamma, 61% (45/74). Concentrations of interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha in MEEs were highly correlated with each other (P < .01 for each association) suggesting increased local production and the expected effects of cytokines stimulating their own production during OM. High concentrations of tumor necrosis factor alpha in MEEs were also associated with a history of multiple placements of tympanostomy tubes (r = .63). CONCLUSIONS: These data suggest a regulatory role for cytokines in inflammation during OM, and suggest that high concentrations of tumor necrosis factor alpha in MEEs may be a marker for OM chronicity.

Effect of detergents on in vitro 7 alpha-hydroxycholesterol formation by rat liver microsomes.

Formation of 7 alpha-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer (GC/MS) operated in selected ion monitoring (SIM) mode. Microsomes from normal rat livers incubated for different periods were found to yield increased 7 alpha-hydroxycholesterol with time. This was also true when incubations contained Tween-80, but in this instance, the rate of 7 alpha-hydroxycholesterol production was lower and dependent on the concentration of Tween used. Similarly, Triton X-100, Renex-30, Kyro EOB, Cutscum, and Emulgen 911 all lowered the formation of 7 alpha-hydroxycholesterol by rat liver microsomes, whereas Triton WR-1339 stimulated its production. Analysis of data obtained from following the enzyme reaction over an extended period using an integrated Michaelis-Menten equation indicated the enzyme possesses a very significant affinity for the product (Ks greater than Kp). Similar analysis shows that Tween-80 is a noncompetitive inhibitor of the enzyme.

Role of serum markers for liver function and liver regeneration in the management of chloroform poisoning.

Accidental or intentional chloroform poisoning is rare, but a few such cases have been reported in literature. We report here a successful management of acute chloroform toxicity in a 33-year-old white female who attempted suicide by injecting one half milliliter of chloroform, followed by drinking half a cup the next morning. Plasma chloroform levels, measured by headspace gas chromatography declined rapidly. Sequential measurement of biomarkers in serum for liver cell necrosis, liver function, and liver regeneration indicated the presence of initial liver damage followed by recovery. These results suggest that in addition to biomarkers for liver cell necrosis, serial determinations of markers for liver regeneration provide objective evidence for recovery from chloroform poisoning and possibly other hepatotoxins.

Experimental alteration of chinchilla middle ear mucosae by bacterial neuraminidase.

Streptococcus pneumoniae secretes a variety of extracellular glycosidase including a neuraminidase which has been found in middle ear effusion from patients with both acute and chronic otitis media. This enzyme cleaves sialic acid from membrane glycoproteins, thereby exposing galactose residues, the penultimate sugar. The ability of partially purified neuraminidase to alter the middle ear mucosa was investigated in the chinchilla. After incubation with neuraminidase, chinchilla middle ears were removed and exposed to galactose residues labeled with tritium. Membrane glycoproteins were solubilized and separated according to molecular weight by sodium dodecylsulfate electrophoresis. Increases in tritium incorporation, when compared to control incubations, indicated that galactose residues had been exposed and sialic acid residues removed from glycoproteins of both high and low molecular weight. Such membrane destruction could contribute significantly to the pathology of otitis media with effusion.

Hydrolase activity in acute otitis media with effusion.

Biochemical studies of middle ear effusions (MEE) from patients with chronic or recurrent otitis media with effusion (OME) have demonstrated the presence of significant levels of certain hydrolytic and oxidative enzymes. We have examined MEE from patients with acute OME for the content of a number of lysosomal hydrolases and find no significant differences in the mean values for acid phosphatase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, hexosaminidase, and neuraminidase between purulent and serous effusions. In every case, the mean activities of these enzymes were greater in culture-positive than in culture-negative effusions although this difference was significant only in the case of neuraminidase. Neuraminidase activity was detected in 78% of those MEEs from which Streptococcus pneumoniae could be cultured and in only 32% to 64% of all other effusions. No correlation was observed between the level of neuraminidase released into the extracellular growth medium and the infectivity of various strains of S pneumoniae.

Lysosomal hydrolases in middle ear effusions.

Biochemical studies of middle ear effusions have demonstrated generally higher levels of certain hydrolytic and oxidative enzymes in mucoid fluids when compared to serous. We have extended these studies by analyzing middle ear effusions for the content of a large number of lysosomal hydrolases. The mean specific activity for alpha-glucosidase in mucoid fluids was found to be ten times that for serous fluids while alpha-mannosidase, beta-glucuronidase, hexosaminidase, acid phosphatase, beta-galactosidase, alkaline phosphatase, and lactate dehydrogenase were found to be three to five times greater in mucoid than serous effusions. In this study the specific enzyme activities for lysosomal hydrolases from purulent effusions were found to be intermediate between the activities in serous and mucoid effusions. No significant correlation was found between the specific activities of lysosomal hydrolases and the presence or absence of bacteria in mucoid or serous middle ear effusions. The hexosaminidase isozyme distribution was found to be identical for serous and mucoid fluids and similar to that found in human serum. However, the isozyme pattern of beta-glucuronidase in mucoid effusions was significantly different than that in normal human serum as mucoid fluids contain a large amount of an anionic isoenzyme of beta-glucuronidase that is barely detectable in human serum.

Neuraminidase activity in middle ear effusions.

Analyses of Streptococcus pneumoniae culture filtrates and middle ear effusions (MEE) containing S pneumoniae for various hydrolytic enzymes have demonstrated substantial levels of neuraminidase activity when measured employing a sensitive fluorometric assay. S pneumoniae neuraminidase exhibits optimum activity near neutral pH (6.0 to 6.5), and catalyzes the cleavage of sialic acid residues from glycoproteins, gangliosides and mucopolysaccharides. S pneumoniae begins secreting large amounts of neutral neuraminidase (mean [means] = 43.3 units/mL culture filtrate) when cells enter the stationary phase. Nearly all (96%) human chronic MEEs yielding positive cultures for S pneumoniae contain neuraminidase activity (means = 0.200 units/mg protein), while only 21.1% to 45.5% of all other effusions contain the enzyme. Middle ear effusions obtained from S pneumoniae infected-chinchillas contained large amounts of neuraminidase activity (approximately 200 units/mL), which decayed exponentially in vivo with an apparent half-life of 8 1/2 days. Three neuraminidase isoenzymes (designated I-III) were identified in S pneumoniae culture filtrates, as well as in MEEs from chinchillas infected with the organism, using a combination of ion-exchange and gel filtration chromatography. With 4-methylumbelliferyl-N-acetylneuraminic acid serving as substrate, preparation I from both culture filtrates and MEEs was characterized by a high Michaelis constant (Km), while forms II and III had low Km values. Preferred substrates were orosomucoid and neuramin-lactose; gangliosides, thyroglobulin, and bovine submaxillary mucin were poorer substrates.

Expression of acute otitis media after receptor blockade of platelet activating factor, thromboxane, and leukotrienes in the chinchilla.

To determine the role of inflammatory products of phospholipid metabolism in acute otitis media (AOM), we infected 128 chinchillas with Streptococcus pneumoniae and randomly assigned them to one of four equal-sized treatment groups receiving intramuscular ampicillin sodium (control) or intramuscular ampicillin plus receptor blockers of platelet activating factor (WEB 2086, 5 mg/d orally), of leukotriene (MK 571, 0.5 mg/d orally), or of thromboxaneA2 (GR 32191B, 5 mg/d orally). All treatments were begun on day 2 postinoculation and continued for 10 days. On days 3, 6, 9, and 12, 8 animals from each group were sacrificed. Effusions were recovered for biochemical assay, and the right middle ears were prepared for histologic study. Differences among groups in the number of ears with effusion or in effusion volume were not statistically significant. In comparison to the control group, mucosal thickness and the number of ears with histopathologic signs of inflammation were significantly less in the GR and WEB treatment groups, but not the MK group. Also, effusion concentrations of free fatty acids, protease, and hydrolytic enzymes were significantly less in those groups. These results show that the addition of a receptor blocker for either platelet activating factor and/or thromboxane to ampicillin in the treatment of AOM reduces mucosal inflammation and decreases the production of other inflammatory chemicals. The failure of a receptor blocker of leukotrienes to moderate disease expression suggests either a less important role for these chemicals in AOM or an insufficient bioavailability of the specific MK 571 inhibitor. These results confirm that platelet activating factor and thromboxane are active mediators of inflammation in AOM.

Upregulation of messenger RNA for inflammatory cytokines in middle ear mucosa in a rat model of acute otitis media.

A rat model for acute otitis media has been established and was used to delineate the temporal expression of messenger RNA for key inflammatory cytokines. Inoculation with live Streptococcus pneumoniae induced a rapid expression of tumor necrosis factor alpha (within 6 hours) followed by upregulation of message for interleukin (IL)-6 (peak at 12 to 24 hours, remaining elevated through 120 hours) and IL-10 (peak at 24 hours). Inducible nitric oxide synthase message was also selectively increased following live bacterial inoculation (peak at 12 to 24 hours). Although there was a detectable inflammatory response to killed bacteria, it was minimal, was of short duration, and preceded the peak for live bacteria; only expression of IL-6 was significantly increased in this group (peak at 12 hours, remaining elevated through 72 hours). We interpret this to be due to an inflammatory response to bacterial products (such as lipopolysaccharide) in the heat-killed bacterial inoculum. The phosphate-buffered saline (PBS)-inoculated group exhibited a transient increase of IL-6 message, which indicates that this cytokine is a sensitive marker of the acute response to trauma. Otherwise, PBS invoked only a slight reaction in the mucosa with respect to the other inflammatory mediators being measured.

Treatment of experimental acute otitis media with ibuprofen and ampicillin.

The efficacy of concurrent treatment of experimental acute otitis media with ibuprofen and ampicillin was evaluated in chinchillas with respect to clearance of the effusion and resolution of mucosal inflammation. Sixty-four chinchillas were infected with Streptococcus pneumoniae and randomly assigned to treatment with either IM ampicillin (control) or ampicillin plus ibuprofen (experimental) beginning on day 2 post inoculation. On days 3, 6, 9 and 12, 8 animals from each group were killed, effusions recovered for biochemical assay and the right middle ears prepared for histological study. Between group differences in the number of ears with effusion and effusion volume were not statistically significant. Mucosal thickness and the frequencies of ears with histopathological signs of inflammation were significantly less in the experimental group when compared to the control group. Differences in the effusion concentrations of total protease, 3 of 4 hydrolytic enzymes and free fatty acids favoring the experimental group were observed at the 6, 9 and 12 day endpoints. Also, at those times the levels of the 3 measured products of the cyclooxygenase pathway were less in the experimental group. These results suggest that the addition of ibuprofen to ampicillin for the treatment of acute otitis media decreases production of select eicosonoids, reduces mucosal inflammation and alters the course of the disease in this model of bacterial infection.

Effect of ibuprofen treatment during experimental acute otitis media.

In this study, the efficacy of concurrent treatment of experimental acute otitis media with ibuprofen and ampicillin was evaluated in chinchillas with respect to clearance of the effusion, presence of mucosal inflammation, and modulation of biochemical markers. Forty chinchillas were infected with non-typable Haemophilus influenzae and randomly assigned to treatment with either IM ampicillin (control) or ampicillin plus ibuprofen beginning on day 2 post inoculation. On days 5 and 10, animals from each group were killed, effusions recovered for biochemical assay, and the middle ears prepared for histological study. Differences in outcome measures favoring the control group were observed at the 5 day endpoint. However, at the 10 day endpoint, mucosal thickness was significantly less, the number of effusion free ears greater, and the concentrations of free fatty acids and thromboxane less in the animals treated with the combined therapy when compared to the control group. These results suggest that the addition of ibuprofen to ampicillin for the treatment of acute otitis media alters disease pathogenesis in this infectious model.