Influence of dietary protein on glomerular filtration before and after bariatric surgery: a cohort study.

BACKGROUND: Obesity-associated elevations in glomerular filtration rate (GFR) are common and may play a role in the development of kidney disease, so identifying the underlying mechanism is important. We therefore studied whether reductions in dietary protein intake, which is known to modulate GFR, explain why GFR decreases after bariatric surgery-induced weight loss. STUDY DESIGN: Cohort study with participants as their own controls. SETTING & PARTICIPANTS: 8 severely obese patients with normal kidney function were recruited from bariatric surgery centers in Indianapolis, IN. All participants were placed on a fixed-protein (50-g/d) diet for 1 week before and after a minimum of a 20-kg weight loss by bariatric surgery and were followed up closely by dieticians for adherence. PREDICTOR: Ad lib versus low-protein diet before versus after bariatric surgery. OUTCOME: Measured GFR, using repeated-measures analysis, was used to estimate the independent effects of diet and surgery. MEASUREMENT: GFR was measured using plasma iohexol clearance. RESULTS: A median of 32.9 (range, 19.5-54.4)kg was lost between the first presurgery visit and first postsurgery visit. Dietetic evaluations and urinary urea excretion confirmed that patients generally adhered to the study diet. GFRs on an ad lib diet were significantly higher before compared to after surgery (GFR medians were 144 (range, 114-178) and 107 (range, 85-147) mL/min, respectively; P=0.01). Although bariatric surgery (-26mL/min; P=0.005) and dietary sodium intake (+7.5mL/min per 100mg of dietary sodium; P=0.001) both influenced GFR, consuming a low-protein diet did not (P=0.7). LIMITATIONS: Small sample size; mostly white women; possible lack of generalizability. CONCLUSIONS: The decrease in GFR observed after bariatric surgery is explained at least in part by the effects of surgery and/or dietary sodium intake, but not by low dietary protein consumption.

Predicting the glomerular filtration rate in bariatric surgery patients.

BACKGROUND/AIMS: Identifying the best method to estimate the glomerular filtration rate (GFR) in bariatric surgery patients has important implications for the clinical care of obese patients and research into the impact of obesity and weight reduction on kidney health. We therefore performed such an analysis in patients before and after surgical weight loss. METHODS: Fasting measured GFR (mGFR) by plasma iohexol clearance before and after bariatric surgery was obtained in 36 severely obese individuals. Estimated GFR was calculated using the Modification of Diet in Renal Disease equation, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation using serum creatinine only, the CKD-EPI equation using serum cystatin C only and a recently derived equation that uses both serum creatinine and cystatin C (CKD-EPIcreat-cystC) and then compared to mGFR. RESULTS: Participants were primarily middle-aged white females with a mean baseline body mass index of 46 ± 9, serum creatinine of 0.81 ± 0.24 mg/dl and mGFR of 117 ± 40 ml/min. mGFR had a stronger linear relationship with inverse cystatin C before (r = 0.28, p = 0.09) and after (r = 0.38, p = 0.02) surgery compared to the inverse of creatinine (before: r = 0.26, p = 0.13; after: r = 0.11, p = 0.51). mGFR fell by 17 ± 35 ml/min (p = 0.007) following surgery. The CKD-EPIcreat-cystC was unquestionably the best overall performing estimating equation before and after surgery, revealing very little bias and a capacity to estimate mGFR within 30% of its true value over 80% of the time. This was true whether or not mGFR was indexed for body surface area. CONCLUSIONS: In severely obese bariatric surgery patients with normal kidney function, cystatin C is more strongly associated with mGFR than is serum creatinine. The CKD-EPIcreat-cystC equation best predicted mGFR both before and after surgery.

Glycated albumin, not hemoglobin A1c, predicts cardiovascular hospitalization and length of stay in diabetic patients on dialysis.

BACKGROUND: The utility of glycated hemoglobin (HbA1c) and glycated albumin (GA) in diabetic dialysis patients remains unknown. GA was previously associated with all-cause hospitalization and patient survival. Relationships between GA, HbA1c, and casual plasma glucose (PG) with cause-specific cardiovascular (CV) disease, infectious disease (ID), and vascular access- (VA) related hospitalization rates and length of stay (LOS) were assessed. METHODS: 444 prevalent diabetic dialysis patients had monthly PG, quarterly GA, and all HbA1c values recorded for 2.33 years; hospitalizations within 17 and 30 days of testing were evaluated. Best-fit, time-dependent Cox models were constructed in unadjusted, case-mix-adjusted (age, sex, race, BMI, diabetes duration, dialysis vintage), and case-mix- plus lab-adjusted (hemoglobin, albumin, phosphorus) models. RESULTS: Mean ± SD diabetes duration was 18.5 ± 10.8 years and dialysis vintage 2.9 ± 2.6 years. In fully adjusted models, CV hospitalization rates were associated with increasing GA (HR 1.32; 95% CI 1.11-1.57; p = 0.002 at 17 days; HR 1.21; p = 0.02 at 30 days) and PG (HR 1.10; 95% CI 1.02-1.17; p = 0.01 at 17 days; HR 1.07; p = 0.03 at 30 days), not HbA1c (HR 1.24; 95% CI 0.89-1.73; p = 0.21 at 17 days; HR 1.26; p = 0.10 at 30 days). LOS for CV admissions was positively associated with GA (HR 1.18; 95% CI 1.01-1.39; p = 0.03), not PG (HR 1.04; 95% CI 0.99-1.10; p = 0.15) or HbA1c (HR 1.03; 95% CI 0.92-1.15; p = 0.21). Admissions due to ID and VA complications (and LOS) did not correlate with these assays. CONCLUSIONS: Improved glycemic control based on GA and PG predicted CV-related hospitalizations; GA also predicted CV hospitalization LOS. HbA1c did not predict cause-specific hospitalizations in dialysis populations.

Independent influence of dietary protein on markers of kidney function and disease in obesity.

Obesity is associated with glomerular hyperfiltration and increased urinary protein excretion, as well as structural and functional changes that lead to kidney disease and failure. Dietary protein mimics obesity's effects on the glomerular filtration rate (GFR) and proteinuria and, in certain circumstances, may have the potential to adversely affect kidney function. Here we tested the hypothesis that dietary protein independently explains elevations in the GFR and proteinuria found in obese persons with a normal serum creatinine. Seventeen patients were randomized in a double-blind, crossover fashion for 1-week periods to high (140 g/day) and low (50 g/day) protein diets with a 1-week washout interval separating these periods. High protein consumption was associated with a very modest but significant increase in the GFR of 5 ± 6 ml/min. Hence, while dietary protein does modulate kidney parameters, it is unlikely to fully account for the elevations in GFR and proteinuria found in obesity.

Capillary electrophoresis of the proteolytic activity of the stratum corneum obtained by tape stripping.

Serine proteases and some cathepsins are present in the stratum corneum. They are known to play a significant role in the pathophysiological mechanism of several dermatological conditions (e.g. atopic dermatitis) and in the induction of itch. Tape stripping of skin is a simple technique used in the investigation of skin barrier function and in the penetration of topically applied drugs. Herein, we show that CE, under stacking conditions, is a well-suited technique to measure the proteolytic activity of enzymes in the stratum corneum. Disks of about 6 mm (id) were cut from adhered tapes and submerged directly in a buffer containing the appropriate peptide substrate. After incubation, the split peptides were separated and detected directly by CE at 214 nm in a borate buffer. The esterase activity on N-benzoyl-tyrosine ethyl ester and the amidase activity on succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and the splitting of hemoglobin were detected by CE. The esterase activity was the highest when compared to the proteolytic activities. Skin scratching increased the enzymatic activity adhered to the tapes. The CE offered over the traditional end-point colorimetric methods the ability to measure the low enzymatic activity and the ability to detect the released peptides directly. This technique is simple, non-invasive, easy to perform and uses non-expensive substrates. It can be useful in quantifying cathepsins and serine proteases in the skin.

Relationship between assays of glycemia in diabetic subjects with advanced chronic kidney disease.

BACKGROUND: Relative to hemoglobin A(1c) (HbA(1c)), glycated albumin (GA) more accurately reflects recent glycemic control in diabetic patients on hemodialysis and peritoneal dialysis. These assays have yet to be compared in patients with advanced chronic kidney disease (CKD). METHODS: HbA(1c) and GA were simultaneously measured in 303 diabetic subjects: 70 with CKD prior to dialysis (CKD-stage 4), 184 with CKD after transplantation (TXP-stage 3) and 49 non-nephropathy controls. RESULTS: Mean estimated GFR was 76, 46 and 26 ml/min in controls, TXP-3 and CKD-4 cases, respectively. Mean (SD) HbA(1c) (%) and GA (%) concentrations were 7.30 (1.40) and 16.8 (4.9) in controls, 7.28 (1.66) and 21.5 (6.4) in CKD-4 cases, and 7.21 (1.62) and 21.2 (5.5) in TXP-3 cases, respectively. The GA:HbA(1c) ratio differed significantly between non-nephropathy controls and both groups of CKD patients (both p < 0.001), but not between CKD-4 and TXP-3 cases (p = 0.92). The glucose:HbA(1c) ratio was inversely associated with GFR in all 254 nephropathy cases (r = -0.13; p = 0.04), while glucose:GA did not vary significantly based upon GFR (r = -0.08; p = 0.24). CONCLUSIONS: The relationship between glycated albumin and HbA(1c) is influenced by the presence of reduced GFR in diabetic patients with CKD. The accuracy of the HbA(1c) assay in diabetic subjects with severe nephropathy requires further investigation, although HbA(1c) performs relatively well with milder CKD.

Measuring the glomerular filtration rate in obese individuals without overt kidney disease.

BACKGROUND: Identifying methods to accurately measure the glomerular filtration rate (GFR) in obese individuals without kidney overt kidney disease is necessary to understanding the pathophysiology and natural history of obesity-related kidney disease. METHODS: Using a cross-sectional design, iohexol clearance and disposition was measured, an optimal sampling schedule was identified, and the reliability of GFR-estimating methods was described in 29 obese individuals with normal serum creatinine levels. Iohexol disposition was measured using population pharmacokinetics. The agreement with GFR-estimating equations was assessed by intraclass coefficients. RESULTS: Mean age was 44 ± 10 years, body mass index 45 ± 10, creatinine 0.7 ± 0.2 mg/dl (62 ± 18 µmol/l) , and cystatin C 0.83 ± 0.18 mg/dl (8.3 ± 1.8 mg/l). Iohexol disposition fit a two-compartment model and 5 sampling windows were identified over a 4-hour period to optimize model accuracy and minimize blood draws. Precision was not compromised with this sampling design. Neither creatinine nor cystatin C were linearly correlated with the measured GFR though cystatin C was independent of body composition. Agreement was fair to poor between the measured GFR and GFR-estimating equations. CONCLUSION: This study offers a rigorous method to study obesity-related kidney disease and improve upon suboptimal GFR-estimating methods.

Enhanced detection in capillary electrophoresis: example determination of serum mycophenolic acid.

In order to overcome the poor absorbency detection limits in CE, two simple strategies were combined to increase the amount of the sample injected: a long capillary to hold extra sample while leaving adequate room for the separation and acetonitrile stacking, which concentrated the sample based on transient pseudo-ITP. The combination of these two strategies yielded sensitivity comparable or better than that of the HPLC with good separation and with better theoretical plate number. The analysis of mycophenolic acid, a common immunosuppressant drug, in serum was used here as an example to illustrate this enhanced detection and its applicability to therapeutic drug monitoring. Acetonitrile was used to remove serum proteins followed by direct injection filling 5-21% of the capillary volume and separation in a borate buffer. The overall CE method compared well to an assay by HPLC as far as sample preparation, correlation coefficient, and especially sensitivity of detection.

Cryoglobulins: an important but neglected clinical test.

Cryoglobulin (CR) denotes a serum immunoglobulin that precipitates at temperatures below 37 degrees C and dissolves on re-warming. CRs are heterogeneous in chemical composition and behave differently in vivo and in vitro. The majority are mixed antigen-antibody complexes that occur with high incidence in autoimmune and infectious disorders. Their measurement is important in the management of patients with vasculitis. CRs elicit variable symptoms in patients, mostly purpura, weakness, and arthralgias, and they require various methods of treatment. Sometimes CRs are not associated with any symptoms; but they can be associated with very severe conditions such as nephropathy and neuropathy. Treatment depends on the symptoms and causes, and on the phenotyping of the CR. Considering the high incidence of CR in diseases such as hepatitis C virus (HCV) infection, together with the high worldwide prevalence of this disease, it is clear that testing for CR is underutilized in clinical practice. CR testing has been neglected in routine clinical laboratories and by clinicians due to several factors, such as the lengthy time for serum CR analysis and failure to appreciate that low levels of CR can be associated with severe symptoms. In a series of 194 serum samples that gave positive tests for CR at our institution, the majority contained low CR concentrations (65% of the samples were type II with a mean of 372 mg/L and 39% of type III with a mean of 216 mg/L; reference range 0-60 mg/L). Case studies are presented to illustrate the importance of such low levels of CR. There is a need for more rapid and more reliable methods for quantification and phenotyping of low concentrations of serum CR. Based on our experience in the routine analysis, quantification, and phenotyping of serum CR, some practical solutions to these problems are presented.

Organic solvent high-field amplified stacking for basic compounds in capillary electrophoresis.

Many water-miscible organic solvents, especially acetonitrile and acetone, bring along significant degrees (approximately 30 times) of stacking by electroinjection through high-field amplified injection for the basic compounds compared to that for aqueous buffers or water. The relative stacking of different compounds in acetonitrile or acetone is different compared to that for water. Stacking by electroinjection in organic solvents is less stringent and easier to accomplish in practice. Acids and salts, in aqueous solutions, can ruin the stacking for both organic and aqueous solvents; however, this effect can be better tolerated by diluting the sample in acetonitrile. Thus, this stacking is termed "organic solvent high-field amplified injection". This stacking by electroinjection is enhanced by increasing the electrophoresis buffer concentration and can be better than that by pressure injection. From the practical aspects, some cationic drugs present in serum such as amiodarone can be detected at the therapeutic levels by electroinjection on the capillary after protein precipitation by acetonitrile.

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis.

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.

Analysis of Tamm-Horsfall protein by high-performance liquid chromatography with native fluorescence.

Tamm-Horsfall (TH) is a large glycoprotein which originates in the kidney and is very abundant in the urine. This protein has been measured mainly by immunoassays. Here we describe a different approach for its measurement based on high-performance liquid chromatography (HPLC) using a molecular exclusion column with native fluorescence detection in the ultraviolet range. This method in addition to measuring the level of the protein has the advantage of detecting changes in size or aggregation. Urine, 1 ml was mixed with 100 microl of 30% NaCl and left at 37 degrees C for 30 min. The urine was centrifuged at 12000 rpm for 20 s. The precipitate was vortex-mixed and dissolved in a triethanolamine buffer. A 20 microl aliquot was injected on a Macrosphere GPC column which was eluted with phosphate buffer and the effluent was detected by a fluorometer set at 280 nm for excitation and 325 nm for emission. Since the protein has a very large molecular mass compared to other urinary and serum proteins we did not experience any interference. It elutes as the first peak (in approximately 2.5 min on a 500 A and 2.7 min on 1000 A). The protein precipitates rapidly < 60 min at 37 degrees C. The detection in the UV is sensitive for this protein down to 1 mg/l in absence of any concentration steps. The method was linear between 1 and 100 mg/l. The R.S.D. was 10.4% (mean 62, n = 10). The mean level in 42 normal individuals was 31 mg/g creatinine and in 30 patients with proteinuria (different renal disorders) was 23 mg/g creatinine.

IgD-kappa myeloma: an unusual case.

A rare case of biclonal IgD-kappa and IgG-kappa myeloma is described. The patient initially presented with anemia, renal insufficiency, and proteinuria. The IgD-kappa, initially, was overlooked as a light chain; however, it decreased in serum concentration after treatment by approximately 90%, in contrast to the IgG-kappa that decreased in serum by approximately 40 % over a 9-yr period. Clinically, the patient responded well to treatment and improved greatly during this period. Practical recommendations are suggested in order to detect such cases.

Basic performance of an enzymatic method for glycated albumin and reference range determination.

BACKGROUND: Glycated albumin (GA) is a medium-term glycemic control marker of diabetes and may be more sensitive to changes in plasma glucose than hemoglobin A1c. We studied where and how many fructosyl groups bind to albumin, and which glycation sites are measured by the enzymatic method for GA. We also studied the basic performance of the enzymatic method for GA. METHODS: Glycated albumin was measured using an enzymatic method (Lucica®GA-L, Asahi Kasei Pharma) on a biochemical autoanalyzer. Molecular weights of purified GA and nonglycated albumin were measured by a mass spectrometry system. Two hundred one healthy volunteers with normal results of oral glucose tolerance testing were recruited to determine the reference range in Americans. RESULTS: The present method measured only glycated amino acids from albumin in serum protein. We estimate that the number of glycated amino acids measured by this method was approximately two per molecule of albumin. The general performance (sensitivity, specificity, reproducibility, linearity, interference) of the method was good. The reference range of GA% in Americans with normal glucose tolerance was determined to be 11.9-15.8% (mean ± 2 standard deviations). Significant differences were not observed between the sexes; however, race differences were observed (higher levels in blacks relative to whites). CONCLUSIONS: The method was specific for measuring glycated amino acids in albumin and had good basic performance characteristics. The reference range in Americans was 11.9-15.8%. This method may be a useful indicator for diabetes control.

Glycated albumin and risk of death and hospitalizations in diabetic dialysis patients.

BACKGROUND AND OBJECTIVES: Relative to hemoglobin (Hb) A(1c), glycated albumin (GA) more accurately reflects glycemic control in patients with diabetes mellitus and ESRD. We determined the association between GA, HbA(1c), and glucose levels with survival and hospitalizations in diabetic dialysis patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Quarterly GA levels were measured for up to 2.33 years in 444 prevalent patients with diabetes and ESRD. Proportional hazard time-dependent covariate models were computed with adjustment for demographic characteristics, comorbidities, and laboratory variables. Similar analyses were performed for available HbA(1c) and monthly random serum glucose determinations. RESULTS: The participants were 53% male, 54% African American, 43% Caucasian, 90% on hemodialysis, with a mean (SD) age of 62 (12) years and median follow-up duration of 2.25 years. GA and HbA(1c) mean ± SD 21.5% ± 6.0%, median 20.4% and mean ± SD 6.9% ± 6.6%, median 1.6%, respectively. There were 156 deaths during the observation period. In best-fit models, predictors of death included increasing GA, increasing age, presence of peripheral vascular disease, decreasing serum albumin, and decreasing hemoglobin concentrations. HbA(1c) and random serum glucose concentrations were not predictive of survival. Increasing GA levels were associated with hospitalization in the 17 days after measurement, whereas HbA(1c) was not. CONCLUSIONS: In contrast to the HbA(1c) and random serum glucose values, GA accurately predicts the risk of death and hospitalizations in patients with diabetes mellitus and ESRD. The GA assay should be considered by clinicians who care for patients with diabetes on dialysis.

Sickle cell trait and development of microvascular complications in diabetes mellitus.

BACKGROUND AND OBJECTIVES: Many African Americans (AA) have both sickle cell trait (SCT) and diabetes mellitus. The objective of this study was to determine whether individuals with diabetes mellitus and SCT have higher rates of microvascular complications relative to those without SCT. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This was a retrospective study comparing albuminuria, estimated GFR (eGFR), and microvascular complications in AA with diabetes on the basis of presence of SCT. The study included 821 outpatients who underwent hemoglobin A1c (HbA1c) testing, and presence of SCT was determined using the HbA1c assay. Medical record review and telephone interviews were performed for AA participants. RESULTS: Data were obtained on 376 AA patients (110 with SCT, 245 with neither SCT nor hemoglobin C trait, and 21 with hemoglobin C trait) and 445 European Americans. The mean eGFR and urinary protein excretion were similar between the three AA subgroups. Analysis revealed that 36.3% of AA nontrait and 22.7% of AA SCT participants had retinopathy, peripheral vascular disease, or end-stage kidney disease (P = 0.01). After adjustment for diabetes duration, age, insulin use, and gender, differences in the prevalence of microvascular complications were not observed. CONCLUSIONS: SCT does not increase the risk of microvascular complications in AA with diabetes mellitus.

Comparison of glycated albumin and hemoglobin A1c concentrations in diabetic subjects on peritoneal and hemodialysis.

BACKGROUND: Relative to hemoglobin A(1c) (HbA(1c)), percentage of glycated albumin (GA%) more accurately reflects recent glycemic control in diabetic hemodialysis (HD) patients. METHODS: To determine the accuracy of glycemic assays in a larger sample including patients on peritoneal dialysis (PD), HbA(1c) and GA% were measured in 519 diabetic subjects: 55 on PD, 415 on HD, and 49 non-nephropathy controls. RESULTS: Mean +/- SD serum glucose levels were higher in HD and PD patients relative to non-nephropathy controls (HD 169.7 +/- 62 mg/dL, PD 168.6 +/- 66 mg/dL, controls 146.1 +/- 66 mg/dL; p = 0.03 HD vs controls, p = 0.13 PD vs controls). GA% was also higher in HD and PD patients (HD 20.6% +/- 8.0%, PD 19.0% +/- 5.7%, controls 15.7% +/- 7.7%; p < 0.02 HD vs controls and PD vs controls). HbA(1c) was paradoxically lower in dialysis patients (HD 6.78% +/- 1.6%, PD 6.87% +/- 1.4%, controls 7.3% +/- 1.4%; p = 0.03 HD vs controls, p = 0.12 PD vs controls). The serum glucose/HbA(1c) ratio differed significantly between dialysis patients and controls (p < 0.0001 HD vs controls, p = 0.002 PD vs controls), while serum glucose/GA% ratio was similar across groups (p = 0.96 HD vs controls, p = 0.64 PD vs controls). In best-fit multivariate models with HbA(1c) or GA% as outcome variable, dialysis status was a significant predictor of HbA(1c) but not GA%. CONCLUSIONS: The relationship between HbA(1c) and GA% differs in diabetic patients with end-stage renal disease who perform either PD or HD compared to those without nephropathy. HbA(1c) significantly underestimates glycemic control in peritoneal and hemodialysis patients relative to GA%.

Rapid quantitation of hemoglobin S in sickle cell patients using the Variant II Turbo analyzer.

A rapid ( approximately 90 sec), fully automated method is described for quantifying hemoglobin S (HbS) by high performance liquid chromatography (HPLC) using the Bio-Rad Variant II Turbo analyzer. Although this instrument is designed to quantify only blood hemoglobin A1c (HbA1c), we show that it can also quantify accurately, without modification, HbS levels in sickle cell patients, provided the blood samples meet certain conditions. The samples should contain detectable hemoglobin F (HbF), but should not contain hemoglobin C (HbC). Under these conditions, blood HbS levels obtained by this method correlate well with those obtained by agarose electrophoresis (r(2) = 0.97, n = 81 patients). We also show that quantitation of blood HbF in sickle cell patients is more accurate by this method than by agarose electrophoresis when the HbF level is in the range from 0.2 to 10%.

A chronic iron-deficient/high-manganese diet in rodents results in increased brain oxidative stress and behavioral deficits in the morris water maze.

Iron deficiency (ID) is especially common in pregnant women and may even persist following childbirth. This is of concern in light of reports demonstrating that ID may be sufficient to produce homeostatic dysregulation of other metals, including manganese (Mn). These results are particularly important considering the potential introduction of the Mn-containing gas additive, methyl cyclopentadienyl manganese tricarbonyl (MMT), in various countries around the world. In order to model this potentially vulnerable population, we fed female rats fed either control (35 mg Fe/kg chow; 10 mg Mn/kg chow) or low iron/high-manganese (IDMn; 3.5 mg Fe/kg chow; 100 mg Mn/kg chow) diet, and examined whether these changes had any long-term behavioral effects on the animals' spatial abilities, as tested by the Morris water maze (MWM). We also analyzed behavioral performance on auditory sensorimotor gating utilizing prepulse inhibition (PPI), which may be related to overall cognitive performance. Furthermore, brain and blood metal levels were assessed, as well as regional brain isoprostane production. We found that treated animals were slightly ID, with statistically significant increases in both iron (Fe) and Mn in the hippocampus, but statistically significantly less Fe in the cerebellum. Additionally, isoprostane levels, markers of oxidative stress, were increased in the brain stem of IDMn animals. Although treated animals were indistinguishable from controls in the PPI experiments, they performed less well than controls in the MWM. Taken together, our data suggest that vulnerable ID populations exposed to high levels of Mn may indeed be at risk of potentially dangerous alterations in brain metal levels which could also lead to behavioral deficits.

Direct injection of organic solvent extracts for capillary electrophoresis.

It is demonstrated here that organic solvents immiscible in water used in sample extraction, such as chloroform, can be injected directly and successfully on the capillary without the need for evaporation and reconstitution. Current continuity was maintained all the time during the run. In order to avoid the rapid evaporation of the organic solvent during the analysis, the aqueous layer was left over the chloroform. This simplified the extraction step, and enabled the injection from the same vial over several hours without dealing with problem of evaporation. The relative peak heights in the electropherograms can be modified by the inclusion in the chloroform of a more polar solvent, by adjusting the pH, or adjusting the salt content of the sample. Addition of a polar solvent to the chloroform improved greatly the precision of the analysis for both the peak height and migration time.