Altered inflammasome activation in neonatal encephalopathy persists in childhood.

Neonatal encephalopathy (NE) is characterized by altered neurological function in term infants and inflammation plays an important pathophysiological role. Inflammatory cytokines interleukin (IL)-1ß, IL-1ra and IL-18 are activated by the nucleotide-binding and oligomerization domain (NOD)-, leucine-rich repeat domain (LRR)- and NOD-like receptor protein 3 (NLRP3) inflammasome; furthermore, we aimed to examine the role of the inflammasome multiprotein complex involved in proinflammatory responses from the newborn period to childhood in NE. Cytokine concentrations were measured by multiplex enzyme-linked immunosorbent assay (ELISA) in neonates and children with NE in the absence or presence of lipopolysaccharide (LPS) endotoxin. We then investigated expression of the NLRP3 inflammasome genes, NLRP3, IL-1ß and ASC by polymerase chain reaction (PCR). Serum samples from 40 NE patients at days 1 and 3 of the first week of life and in 37 patients at age 4-7 years were analysed. An increase in serum IL-1ra and IL-18 in neonates with NE on days 1 and 3 was observed compared to neonatal controls. IL-1ra in NE was decreased to normal levels at school age, whereas serum IL-18 in NE was even higher at school age compared to school age controls and NE in the first week of life. Percentage of LPS response was higher in newborns compared to school-age NE. NLRP3 and IL-1ß gene expression were up-regulated in the presence of LPS in NE neonates and NLRP3 gene expression remained up-regulated at school age in NE patients compared to controls. Increased inflammasome activation in the first day of life in NE persists in childhood, and may increase the window for therapeutic intervention.

Folinic acid improves verbal communication in children with autism and language impairment: a randomized double-blind placebo-controlled trial.

We sought to determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment in a double-blind placebo control setting. Forty-eight children (mean age 7 years 4 months; 82% male) with ASD and language impairment were randomized to receive 12 weeks of high-dose folinic acid (2 mg kg-1 per day, maximum 50 mg per day; n=23) or placebo (n=25). Children were subtyped by glutathione and folate receptor-α autoantibody (FRAA) status. Improvement in verbal communication, as measured by a ability-appropriate standardized instrument, was significantly greater in participants receiving folinic acid as compared with those receiving placebo, resulting in an effect of 5.7 (1.0,10.4) standardized points with a medium-to-large effect size (Cohen's d=0.70). FRAA status was predictive of response to treatment. For FRAA-positive participants, improvement in verbal communication was significantly greater in those receiving folinic acid as compared with those receiving placebo, resulting in an effect of 7.3 (1.4,13.2) standardized points with a large effect size (Cohen's d=0.91), indicating that folinic acid treatment may be more efficacious in children with ASD who are FRAA positive. Improvements in subscales of the Vineland Adaptive Behavior Scale, the Aberrant Behavior Checklist, the Autism Symptom Questionnaire and the Behavioral Assessment System for Children were significantly greater in the folinic acid group as compared with the placebo group. There was no significant difference in adverse effects between treatment groups. Thus, in this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.

Advancing the Sustainability of US Agriculture through Long-Term Research.

Agriculture in the United States must respond to escalating demands for productivity and efficiency, as well as pressures to improve its stewardship of natural resources. Growing global population and changing diets, combined with a greater societal awareness of agriculture's role in delivering ecosystem services beyond food, feed, fiber, and energy production, require a comprehensive perspective on where and how US agriculture can be sustainably intensified, that is, made more productive without exacerbating local and off-site environmental concerns. The USDA's Long-Term Agroecosystem Research (LTAR) network is composed of 18 locations distributed across the contiguous United States working together to integrate national and local agricultural priorities and advance the sustainable intensification of US agriculture. We explore here the concept of sustainable intensification as a framework for defining strategies to enhance production, environmental, and rural prosperity outcomes from agricultural systems. We also elucidate the diversity of factors that have shaped the past and present conditions of cropland, rangeland, and pastureland agroecosystems represented by the LTAR network and identify priorities for research in the areas of production, resource conservation and environmental quality, and rural prosperity. Ultimately, integrated long-term research on sustainable intensification at the national scale is critical to developing practices and programs that can anticipate and address challenges before they become crises.

Ability of radial arterial palpation and observation of the pulse oximetry trace to estimate non-invasive systolic pressure in healthy volunteers and in women undergoing spinal anaesthesia for elective caesarean section.

We assessed the ability of palpating the radial arterial pulse and observing the oximeter trace to estimate the automated non-invasive systolic pressure reading in 20 healthy female volunteers and 20 parturients undergoing spinal anaesthesia for elective caesarean section. Using real-time values of cuff pressure during inflation/deflation, the pressure was recorded when the manually palpated radial arterial pulse or pulse oximeter waveform disappeared and reappeared. The actual measured systolic pressure was noted and the results compared using Bland-Altman analysis. In the volunteers, the bias/precision for radial arterial palpation was -12.9/22.1 mmHg (inflation) and -9.7/16.7 mmHg (deflation), and for oximetry 29.5/18.8 mmHg (inflation) and -20.7/21.7 mmHg (deflation). In the parturients, the bias/precision was -19.0/47.6 mmHg (inflation) and -15.5/51.0 mmHg (deflation) for arterial palpation, and 22.6/16.1 mmHg (inflation) and -14.2/19.9 mmHg (deflation) for oximetry. Our results suggest that neither method is accurate at estimating the non-invasive systolic pressure, with all except oximetry (inflation) underestimating it by approximately 10-20 mmHg and with poor precision.

Nitrogen and phosphorus runoff losses from variable and constant intensity rainfall simulations on loamy sand under conventional and strip tillage systems.

Further studies on the quality of runoff from tillage and cropping systems in the southeastern USA are needed to refine current risk assessment tools for nutrient contamination. Our objective was to quantify and compare effects of constant (Ic) and variable (Iv) rainfall intensity patterns on inorganic nitrogen (N) and phosphorus (P) losses from a Tifton loamy sand (Plinthic Kandiudult) cropped to cotton (Gossypium hirsutum L.) and managed under conventional (CT) or strip-till (ST) systems. We simulated rainfall at a constant intensity and a variable intensity pattern (57 mm h(-1)) and collected runoff continuously at 5-min intervals for 70 min. For cumulative runoff at 50 min, the Iv pattern lost significantly greater amounts (p < 0.05) of total Kjeldahl N (TKN) and P (TKP) (849 g N ha(-1) and 266 g P ha(-1) for Iv; 623 g N ha(-1) and 192 g P ha(-1) for Ic) than did the Ic pattern. However, at 70 min, no significant differences in total losses were evident for TKN or TKP from either rainfall intensity pattern. In contrast, total cumulative losses of dissolved reactive P (DRP) and NO3-N were greatest for ST-Ic, followed by ST-Iv, CT-Ic, and CT-Iv in diminishing order (69 g DRP ha(-1) and 361 g NO3-N ha(-1); 37 g DRP ha(-1) and 133 g NO3-N ha(-1); 3 g DRP ha(-1) and 58 g NO3-N ha(-1); 1 g DRP ha(-1) and 49 g NO3-N ha(-1)). Results indicate that constant-rate rainfall simulations may overestimate the amount of dissolved nutrients lost to the environment in overland flow from cropping systems in loamy sand soils. We also found that CT treatments lost significantly greater amounts of TKN and TKP than ST treatments and in contrast, ST treatments lost significantly greater amounts of DRP and NO3-N than CT treatments. These results indicate that ST systems may be losing more soluble fractions than CT systems, but only a fraction the total N (33%) and total P (11%) lost through overland flow from CT systems.

Variable rainfall intensity and tillage effects on runoff, sediment, and carbon losses from a loamy sand under simulated rainfall.

The low-carbon, intensively cropped Coastal Plain soils of Georgia are susceptible to runoff, soil loss, and drought. Reduced tillage systems offer the best management tool for sustained row crop production. Understanding runoff, sediment, and chemical losses from conventional and reduced tillage systems is expected to improve if the effect of a variable rainfall intensity storm was quantified. Our objective was to quantify and compare effects of a constant (Ic) intensity pattern and a more realistic, observed, variable (Iv) rainfall intensity pattern on runoff (R), sediment (E), and carbon losses (C) from a Tifton loamy sand cropped to conventional-till (CT) and strip-till (ST) cotton (Gossypium hirsutum L.). Four treatments were evaluated: CT-Ic, CT-Iv, ST-Ic, and ST-Iv, each replicated three times. Field plots (n=12), each 2 by 3 m, were established on each treatment. Each 6-m2 field plot received simulated rainfall at a constant (57 mm h(-1)) or variable rainfall intensity pattern for 70 min (12-run ave.=1402 mL; CV=3%). The Iv pattern represented the most frequent occurring intensity pattern for spring storms in the region. Compared with CT, ST decreased R by 2.5-fold, E by 3.5-fold, and C by 7-fold. Maximum runoff values for Iv events were 1.6-fold higher than those for Ic events and occurred 38 min earlier. Values for Etot and Ctot for Iv events were 19-36% and 1.5-fold higher than corresponding values for Ic events. Values for Emax and Cmax for Iv events were 3-fold and 4-fold higher than corresponding values for Ic events. Carbon enrichment ratios (CER) were or=1.0 for CT plots (except for first 20 min). Maximum CER for CT-Ic, CT-Iv, ST-Ic, and ST-Iv were 2.0, 2.2, 1.0, and 1.2, respectively. Transport of sediment, carbon, and agrichemicals would be better understood if variable rainfall intensity patterns derived from natural rainfall were used in rainfall simulations to evaluate their fate and transport from CT and ST systems.

Differential Cell Composition and Cytokine Expression Within Lymph Node Granulomas from BCG-Vaccinated and Non-vaccinated Cattle Experimentally Infected with Mycobacterium bovis.

Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunization of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterize the differences in the immune response following M. bovis infection between BCG-vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stages III and IV granulomas was reduced. IHC using cell surface markers revealed the number of CD68+ (macrophages), CD3+ (T lymphocytes) and WC1+ cells (γδ T cells) to be significantly reduced in lymph node granulomas of BCG-vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG-vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFß expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG-vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNF-α demonstrated significantly increased expression within the lymph node granulomas of BCG-vaccinated cattle. This is suggestive of a protective role for IFN-γ and TNF-α in response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine can reduce the number and severity of lesions, induce a different phenotypic response and increase the local expression of key cytokines related to protection.

Contribution of subunits to the function of luteinizing hormone/human chorionic gonadotropin recombinants.

The alpha- and beta-subunits of hCG and of ovine and porcine LH were used to prepare all nine homologous and heterologous alpha beta-recombinants. EAch purified recombinant was assayed in vitro, using dispersed Leydig tumor cells derived from the M5480P tumor, for its ability to stimulate steroidogenesis and to inhibit [125I]iodo-hCG binding. It was found that the potency of a given recombinant in both assays was most similar to that of the hormone from which the beta-subunit was derived. For example, hCG and hCG beta-containing recombinants were invariably more potent than LH and LH beta-containing recombinants. However, within groups of recombinants containing a common beta-subunit, the alpha-subunit exhibited modulatory effects on the biological potencies. The different observed potencies did not result from alpha beta dissociation since the recombinants were stable in dilute solution at 37 C for periods greatly exceeding that of the assay conditions. LH and the LH beta-containing recombinants were found to dissociate more readily from the Leydig tumor cell gonadotropin receptor than hCG and hCG beta-containing recombinants. (These experiments were performed under conditions where internalization was minimal.) However, analogous to the potency measurements, the alpha-subunit contributed to the rate of dissociation. For example, in recombinants with a common beta-subunit, hCG alpha conferred the greatest stability to the hormone-receptor interaction. These results emphasize a positive relationship between receptor occupancy and biological potency. Whereas the beta-subunit of these gonadotropins seems to exhibit the predominant influence in determining potency, it is clear that both subunits contribute to biological activity. This could involve direct effects as well as induced conformational changes in the complementary subunit.

Glycosylation of ovine prolactin during cell-free biosynthesis.

Recently, a glycosylated form of ovine PRL (oPRL) was isolated from a crude pituitary preparation. As glycosylation of PRL was unexpected and because the composition of the oligosaccharide-containing peptide indicated the carbohydrate portion to be extensively degraded, studies of the glycosylation of oPRL during cell-free biosynthesis were initiated. Two glycosylated forms of oPRL can be recognized when biosynthesis occurs in ovine pituitary microsomes. Both forms are converted to mature PRL by digestion with endoglycosidase H and, thus, appear to contain only asparagine-linked, high mannose-type carbohydrate moieties. In contrast, immunoprecipitates from bovine pituitary microsomes consist of the expected (and nonglycosylated) pre-PRL and PRL. The results are consistent with the absence of a sequence segment in the bovine hormone which permits glycosylation (Asn-X-Ser- or Thr-) and the presence of the segment Asn31-Leu-Ser- in the ovine hormone. The occurrence of two glycosylated forms of oPRL is not understood; it may result from an additional site in oPRL capable of glycosylation.

Alpha-subunit conformation in glycoprotein hormones and recombinants as assessed by specific antisera.

RIAs specific for the alpha-subunit of glycoprotein hormones were used to monitor the subunit conformation in ovine FSH and TSH and in recombinants of ovine LH alpha with (ovine and porcine) LH beta and hCG beta. Differences in log-logit slopes of the RIA dilution curves were interpreted to indicate changes, presumably conformational, in the local environment of the antigenic determinants of the alpha-subunit. In all but one case, free ovine LH alpha yielded a slope that was distinct from those of the hormones and recombinants. FSH, TSH, and the homologous LH recombinant all exhibited different slopes, and the recombinant ovine LH alpha-hCG beta was characterized by a slope that was distinct from the identical slopes of the recombinants ovine LH alpha-ovine LH beta and ovine LH alpha-porcine LH beta. These results suggest that a conformational change occurs in the alpha-subunit upon association with a beta-subunit and that different beta-subunits may induce distinct conformations in a common alpha-subunit.

Rapid and easy separation of the subunits of bovine and human glycoprotein hormones by use of high performance liquid chromatography.

A single method of reverse phase high performance liquid chromatography is used to separate the subunits of human and bovine glycoprotein hormones. This rapid and easy method is applicable for the separation and detection of subunits from as little as 10 micrograms hormone or the isolation of subunits from as much as 100 mg hormone. Separation is achieved by chromatography on a Vydac 218TP1010 column with a linear (60-min) gradient of 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide to a solvent containing 50% acetonitrile and 50% 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide. Although in some cases the interaction between the hydrophobic support and the hormone is sufficient for dissociation, preincubation of the hormone with guanidine hydrochloride ensures optimum dissociation and improves resolution of the subunits. The subunits isolated by high performance liquid chromatography are functional in that they will reassociate with their counterpart subunits.

TSH crosslinks to the TSH receptor through the beta subunit.

TSH binds specifically to the TSH receptor present on the surface of thyroid follicular cells and consequently activates adenylate cyclase. It was not known, however, which of the two subunits of the TSH bound to the receptor. By covalently crosslinking TSH to its receptor and characterizing the product with antisera specific for either the alpha or beta subunit of TSH, we have shown that the TSH beta subunit but not the TSH alpha subunit crosslinks to the TSH receptor.

Comparison of lithium ratio between African-American and Caucasian bipolar patients.

Lithium RBC/plasma ratio (LR) was studied in 34 bipolar subjects on therapeutic doses of lithium carbonate. The sample was divided into 22 Caucasians and 12 African-Americans to observe possible ethnic differences in LR as previously reported. The latter group demonstrated a higher LR as well as increased reports of side effects (p < .05), even after controlling previous confounding factors. Our findings suggest that African-Americans may be more susceptible to the side effects associated with lithium treatment, and consequently, lower dosages may be necessary for this group.

Gender effects on persistent cerebral metabolite changes in the frontal lobes of abstinent cocaine users.

OBJECTIVE: Previous studies found functional changes in the frontal brain region and regions with projections to the frontal lobe in cocaine users. The aim of this study was to investigate persistent neurochemical changes in the frontal lobes of subjects with a history of crack cocaine dependence and to determine whether these changes are different in male and female users. METHOD: The frontal gray and white matter of 64 young asymptomatic and abstinent (> 5 months) cocaine users (34 male and 30 female) and 58 healthy comparison subjects without a history of drug abuse was evaluated with localized proton magnetic resonance spectroscopy (1H-MRS). RESULTS: Two-way analysis of variance showed significant cocaine effects on the concentration of frontal gray matter N-acetyl compounds, on the ratio of frontal white matter N-acetyl compounds to creatine levels, on frontal gray and white matter myoinositol levels, and on the ratio of myoinositol to creatine. Significant gender effects were observed for frontal gray matter choline-containing compounds, the ratio of choline-containing compounds to creatine, and the percentage of CSF in both gray and white matter. Interaction effects of cocaine and gender were observed for creatine, N-acetyl/creatine ratio, and myoinositol/creatine ratio in frontal white matter. CONCLUSIONS: Cocaine use is associated with neuronal injury (with decreased N-acetyl compounds) in the frontal cortex and glial activation (with increased myoinositol) in both frontal gray and white matter. In the frontal lobe, cocaine affects male users differently than female users. Future studies on the effects of cocaine abuse should control for the effects of gender-specific neurotoxicity.

Cocaine use, hypertension, and end-stage renal disease.

Cocaine use has been associated with both acute renal failure and hypertension (HTN), but only recently have data suggested it may lead to a chronic insidious form of renal failure. We designed a cross-sectional study to compare the association of cocaine use in hemodialysis patients with and without a diagnosis of HTN-related end-stage renal disease (HTN-ESRD). Two hundred one black patients from two outpatient hemodialysis units in an urban community were evaluated. There were 193 eligible patients, 106 men and 87 women, aged 49.28 +/- 14.4 years. A history of significant cocaine use before dialysis was reported by 55 of 193 subjects (28.5%). A diagnosis of HTN-ESRD was reported in 49 of 55 cocaine users (89.1%) compared with 64 of 138 nonusers (46.38%; odds ratio, 9.44; 95% confidence interval, 3.79 to 23.49; P < 0.0005). Of the 113 subjects with HTN-ESRD, 49 subjects (43.4%) had a history of cocaine abuse, either alone or in combination with other drugs. Subjects with HTN-ESRD with cocaine use were younger than those without cocaine use (40.7 +/- 9.0 versus 53.8 +/-15.3 years; P < 0.0005) and had a shorter reported duration of HTN (5.3 +/- 5.4 versus 12.7 +/- 9.8 years; P < 0.0005, adjusted for age and sex). In our urban dialysis population, a clinical diagnosis of HTN-ESRD was strongly associated with a history of cocaine use and earlier onset of ESRD. Cocaine should be considered as a cause of ESRD in patients without a clear cause of renal failure.

Characterization and biological effects of recombinant human erythropoietin.

Human recombinant erythropoietin (rHuEPO) has been purified to apparent homogeneity and compared to purified human urinary erythropoietin (EPO). Both the purified natural and recombinant EPO preparations were characterized in a competition radioimmunoassay (RIA), the exhypoxic polycythemic mouse bioassay, in vitro tissue culture bioassays using bone marrow cells, and by Western analysis. In the immunological and biological activity assays, the rHuEPO shows a dose response which parallels that of the natural hormone. By Western analysis, the recombinant and human urinary EPO migrate identically. Administration of rHuEPO increases the hematocrit of normal mice in a dose-dependent manner. Additionally, the rHuEPO is able to increase the hematocrit of rats made uremic as a result of subtotal nephrectomy. In summary, by all criteria examined, the rHuEPO is biologically active and equivalent to the natural hormone.

Estrogen protects primary cortical neurons from glutamate toxicity.

The gonadal steroid estrogen has been shown to affect neuronal growth, differentiation and survival. We examined the ability of estrogen to protect primary cortical neurons from toxicity induced by the excitatory neurotransmitter glutamate. In these experiments, a 24-h pretreatment with 15 and 50 nM 17 beta-estradiol significantly reduced cellular lactate dehydrogenase (LDH) release from primary cortical neurons, indicating that neurons treated with 17 beta-estradiol were protected from a toxic glutamate exposure. Pretreatment with related steroids such as progesterone, dihydrotestosterone, dexamethasone or cholesterol did not significantly decrease LDH release. The anti-estrogen tamoxifen blocked the protective effects of 17 beta-estradiol suggesting that a classical steroid hormone receptor may be involved in the mechanism subserving estrogen neuroprotection during glutamate toxicity.

The utility of routine frozen section examination for intraoperative diagnosis of thyroid cancer.

BACKGROUND: The value of routine frozen section examination for intraoperative diagnosis of thyroid cancer and determination of extent of thyroidectomy is controversial and needs to be evaluated on an institution to institution basis. METHODS: A prospective evaluation of 76 patients with nodular thyroid disease who had an adequate fine needle aspiration biopsy (FNAB) underwent thyroidectomy with routine thyroid frozen section examination. A direct comparison of FNAB and frozen section examination, along with a cost benefit analysis of frozen section examination, was completed. RESULTS: The thyroid pathology was carcinoma in 24, follicular adenoma in 24, multinodular goiter in 24, thyroiditis in 3, and a cyst in 1 patient. The sensitivity, specificity, and accuracy of frozen section examination were 93%, 100%, and 97%, respectively, compared with 88%, 89%, and 91% for FNAB (P > 0.05). Diagnosis was deferred in 38 patients (50%) in whom frozen section examination showed a follicular neoplasm. One to 6 frozen section examinations were obtained per patient with alteration in intraoperative management in only 2 patients (3%) at a charge of $246 to $606 per patient and a total charge of $26,040. CONCLUSION: In patients with an adequate FNAB, frozen section examination rarely affected intraoperative decision making in thyroid surgery and its routine use was not cost effective.

The role of fine-needle aspiration biopsy and flow cytometry in the evaluation of persistent neck adenopathy.

BACKGROUND: The role of fine-needle aspiration biopsy (FNAB) and flow cytometry (FCM) in the evaluation of persistent neck adenopathy has not been completely defined. METHODS: All patients who underwent FNAB of an abnormal lymph node from 1990 to 1997 were identified, and the results of FNAB and FCM were correlated with histology or clinical follow-up. RESULTS: FNAB was performed on 133 patients with an abnormal lymph node in the neck. FNAB was malignant in 82 (62%), atypical in 13 (10%), benign in 27 (20%), and nondiagnostic in 11 (8%) patients. FNAB was compared with histologic results in 114 (86%) or with the results of clinical follow-up (average 17 months) in 12 patients (9%). Seven patients (5%) were lost to follow-up. Lymphoma and metastatic carcinoma were diagnosed in 25 (19%) and 76 (57%) patients, respectively. Sensitivity of FNAB and FCM was 96% and 100%, respectively, and specificity was 58% and 100%, respectively. Patients with Hodgkin's lymphoma accounted for 80% of false negative FCM results. FCM was nondiagnostic in 3 of 34 patients (9%). Definitive subclassification of lymphoma was accomplished in only 3 of 17 (18%) patients in whom FCM was performed. CONCLUSIONS: FNAB is reliable in identifying metastatic disease, non-Hodgkin's lymphoma, and reactive lymphadenopathy in the neck. FNAB and FCM still have a limited role in definitive diagnosis of Hodgkin's lymphoma and classification of non-Hodgkin's lymphoma, underscoring the importance of excisional biopsy.

Densimetric determination of carbohydrate content in glycoproteins.

Carbohydrates play important roles in activity, stability and pharmacokinetics of glycoproteins and the degree of glycosylation varies with proteins. In this communication, a simple method of determining the carbohydrate content was developed, which consists of measuring the density increments of a glycoprotein and its non-glycosylated counterpart, and then dividing the difference between the two values by the density increment of carbohydrates. The density increment was relatively constant for various sugars except for sialic acid, and hence assumed to be 0.39. Thus, we obtained carbohydrate contents of 38, 28, 8 and 7% for Chinese hamster ovary cell-expressed erythropoietin (EPO), stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and platelet-derived growth factor (PDGF), respectively. These values are in close agreement with those determined by other methods.