The impact of sickle cell trait on glycated haemoglobin in diabetes mellitus.

AIMS: To determine the effect of sickle cell trait on measurement of glycated haemoglobin (HbA(1c)) in African American patients with diabetes mellitus. METHODS: This is a retrospective study including 885 outpatients who underwent HbA(1c) testing. Medical record review and sickle cell trait determinations based on the HbA(1c) assay were performed in African American participants. The relationship between HbA(1c) and serum glucose measurements was analysed. RESULTS: Data were obtained from 385 AA (109 with SCT, 22 with haemoglobin C trait and 254 without haemoglobinopathy) and 500 European American patients. In a model created through multivariate repeated-effects regression, the relationship between HbA(1c) and simultaneous serum glucose did not differ between African American subjects with and without the sickle cell trait, but differed between African American subjects without the sickle cell trait and European Americans (P = 0.0002). CONCLUSIONS: Sickle cell trait does not impact the relationship between HbA(1c) and serum glucose concentration. In addition, it does not appear to account for ethnic difference in this relationship between African Americans and whites.

Ethnic variation in the correlation between random serum glucose concentration and glycated haemoglobin.

AIMS: To determine if the relationship between serum glucose concentration and glycated haemoglobin is different between African-Americans and whites. METHODS: Retrospective cross-sectional study comparing the association between glycated haemoglobin and serum glucose levels, based upon ethnicity. Two databases were evaluated: (i) 4215 African-American and 6359 white outpatients who had simultaneous glycated haemoglobin, random serum glucose and creatinine concentration measurements between 2000 and 2007 at the North Carolina Baptist Hospital and (ii) 1021 white and 312 African-American Diabetes Heart Study (DHS) participants. RESULTS: In North Carolina Baptist Hospital clinic attendees, a given glycated haemoglobin was associated with higher serum glucose concentrations in African-Americans compared with whites. In a multivariate model with glycated haemoglobin as the outcome variable, racial differences remained significant after adjustment for serum glucose, age, gender and kidney function. For individuals with a serum glucose between 5.6 and 8.3 mmol/l, the glucose : glycated haemoglobin ratio was 1.03 +/- 0.16 mmol/l/% in white individuals and 0.99 +/- 0.17 mmol/l/% in African-Americans (P < 0.0001). For a glycated haemoglobin value of 7.0%, there was a 0.98-mmol/l difference in predicted serum glucose concentration in 50-year-old African-American men, relative to white. Results were replicated in the DHS, where in a best-fit linear model, after adjustment for glucose, African-American race was a significant predictor of glycated haemoglobin (P < 0.0001). CONCLUSIONS: African-Americans have higher glycated haemoglobin values at given serum glucose concentrations relative to whites. This finding may contribute to the observed difference in glycated haemoglobin values reported between these race groups.

Comparison of glycated albumin and hemoglobin A(1c) levels in diabetic subjects on hemodialysis.

Glycated albumin is thought to more accurately reflect glycemic control in diabetic hemodialysis patients than hemoglobin A(1c) because of shortened red cell survival. To test this, glycated hemoglobin and albumin levels were measured in blood samples collected from 307 diabetic subjects of whom 258 were on hemodialysis and 49 were without overt renal disease. In diabetic subjects with renal disease, relative to those without, the mean serum glucose and glycated albumin concentrations were significantly higher while hemoglobin A(1c) tended to be lower. The glycated albumin to hemoglobin A(1c) ratio was significantly increased in dialysis patients compared with the controls. Hemoglobin A(1c) was positively associated with hemoglobin and negatively associated with the erythropoietin dose in hemodialysis patients, whereas these factors and serum albumin did not significantly impact glycated albumin levels. Using best-fit multivariate models, dialysis status significantly impacted hemoglobin A(1c) levels without a significant effect on glycated albumin. Our results show that in diabetic hemodialysis patients, hemoglobin A(1c) levels significantly underestimate glycemic control while those of glycated albumin more accurately reflect this control.

Analysis of the antiepileptic drug keppra by capillary electrophoresis.

A simple and rapid method for determination of the new antiepileptic drug keppra (levetiracetam) by capillary electrophoresis in borate buffer containing sodium dodecyl sulfate is described. The serum was injected without any treatment. The method compared well to high performance liquid chromatography. The mean of keppra in the serum of 35 patients was 25 mg/l (range 7-77 mg/l).

Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy.

INTRODUCTION: Medullary cystic kidney disease 2 (MCKD2) and familial juvenile hyperuricaemic nephropathy (FJHN) are both autosomal dominant renal diseases characterised by juvenile onset of hyperuricaemia, gout, and progressive renal failure. Clinical features of both conditions vary in presence and severity. Often definitive diagnosis is possible only after significant pathology has occurred. Genetic linkage studies have localised genes for both conditions to overlapping regions of chromosome 16p11-p13. These clinical and genetic findings suggest that these conditions may be allelic. AIM: To identify the gene and associated mutation(s) responsible for FJHN and MCKD2. METHODS: Two large, multigenerational families segregating FJHN were studied by genetic linkage and haplotype analyses to sublocalise the chromosome 16p FJHN gene locus. To permit refinement of the candidate interval and localisation of candidate genes, an integrated physical and genetic map of the candidate region was developed. DNA sequencing of candidate genes was performed to detect mutations in subjects affected with FJHN (three unrelated families) and MCKD2 (one family). RESULTS: We identified four novel uromodulin (UMOD) gene mutations that segregate with the disease phenotype in three families with FJHN and in one family with MCKD2. CONCLUSION: These data provide the first direct evidence that MCKD2 and FJHN arise from mutation of the UMOD gene and are allelic disorders. UMOD is a GPI anchored glycoprotein and the most abundant protein in normal urine. We postulate that mutation of UMOD disrupts the tertiary structure of UMOD and is responsible for the clinical changes of interstitial renal disease, polyuria, and hyperuricaemia found in MCKD2 and FJHN.

Multisite evaluation of a new dipstick for albumin, protein, and creatinine.

The goal of our study was to perform a multisite evaluation of a new urine dipstick called Multistix PROtrade mark (Bayer, Elkhart, IN), which has reagent pads for the simultaneous assay of urinary albumin, protein, and creatinine. Patients' urine specimens were assayed at four sites with these dipsticks and with the familiar Bayer Multistix 10SG dipsticks for protein. The new dipstick pads for albumin are impregnated with bis (3',3"-diiodo-4',4"-dihydroxy-5',5"-dinitrophenyl)-3,4,5,6-tetrabromo-sulfonephthalein (DIDNTB) dye. These dipsticks also have a novel pad that estimates urinary creatinine using the peroxidase activity of the copper-creatinine complex. We determined the interlaboratory agreement of these dipsticks by comparing dipstick results to values obtained by quantitative analytical methods. We found that dividing the dipsticks' albumin or protein results by the creatinine concentration reduced the number of false-positive albumin or protein values observed in concentrated urines, and reduced the number of false negatives in dilute urines. The ratio of albumin to creatinine, or protein to creatinine gives a better measure of albumin or protein excretion. Compared to reading by eye, the dipstick results agreed better with the quantitative assays when they were read by a reflectometer (Bayer Clinitek).

Albuminuria and proteinuria in hospitalized patients as measured by quantitative and dipstick methods.

We tested patients' urines for albumin, protein, and creatinine by quantitative and dipstick methods. The concentrations of these analytes were established by quantitative, cuvet-based chemistry methods that we assumed gave the "correct" values. There was good to excellent agreement of the dipstick results with the quantitative methods for the above three analytes. We found many patients who excreted pathological amounts of albumin and/or protein who did not have a diagnosis of kidney disease or other likely causes of proteinuria, suggesting that albuminuria and/or proteinuria were underdiagnosed in our group of patients. Those with cardiovascular disease, kidney disease, or diabetes showed the greatest predictive value of a positive test for albumin or protein by dipstick. Dipstick testing for albumin, protein, and creatinine had good or excellent agreement with quantitative methods. The dipstick tests were easy to use, simple, and low in cost, and can serve well for point-of-care testing.

Analysis of glutathione by capillary electrophoresis based on sample stacking.

Glutathione is a small peptide, which participates in cellular oxidation-reduction and detoxification. It is present in most biological tissues at different concentrations. The oxidized and reduced forms of the peptide were measured in erythrocytes and myocardial tissue by capillary electrophoresis based on stacking. After tissue homogenization or hemolysis of the red blood cells, the samples were deproteinized with acetonitrile and injected filling about 13% of the capillary volume. The electrophoresis was performed at 10 kV using a separation buffer of 250 mM borate, 50 mM Tris, pH 8.0. Sample stacking increased the sensitivity of detection by 10-20-fold.

Decreasing the variability observed in urine analysis.

Urine analysis is affected significantly by biological variability. The objective of this study was to study the feasibility of reducing the biological variability of excretion of various analytes in urine, especially albumin in children with diabetes, by mixing small volumes of early morning samples. Twenty-two male children with type 1 diabetes collected early morning aliquots of approximately 10 ml of urine on 3 consecutive days and kept them refrigerated in sealed containers. The urine collection was repeated every 4-6 months in the diabetic children. Ten normal children and 10 normal adults participated as controls. The specimens were analyzed individually and as mixed samples for each subject. Mixing the 3 urine samples before analysis decreased the biological variability of all urine assays (albumin, glucose, creatinine, total protein, potassium). The diabetic children had 3 times higher variability of urine albumin (as a ratio to creatinine) compared to normal children, when the urine samples were collected individually (61% vs 19%, respectively). The variability in the diabetic children decreased when the 3 specimens were analyzed as a single sample after mixing, especially when urine albumin was expressed as a ratio to creatinine. Blood glycated hemoglobin levels correlated better with urine glucose levels when 3 urine samples were mixed before analysis.

Stacking and discontinuous buffers in capillary zone electrophoresis.

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.

Hemoglobin A2 quantification by capillary zone electrophoresis.

Hemoglobin A2 (HbA2) comprises about 2.2% of the total hemoglobin in the erythrocytes. The separation and quantitation of this minor hemoglobin by capillary electrophoresis (CE) using an arginine Tris buffer is described. Some of the variables affecting the accuracy and precision of HbA2 quantification are investigated. Furthermore, the quantification of this hemoglobin by CE is compared to that of a microcolumn chromatography method. The CE method is better suited than the microcolumn method for measuring HbA2 in the sickle cell trait.

Stacking in capillary zone electrophoresis.

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.

Field amplified injection in the presence of salts for capillary electrophoresis.

Salts in the sample are detrimental to the stacking by the field-amplified injection. However, physiological samples often contain salts at levels of about 1% which can diminish the peak height or cause band spreading instead of stacking. Using different analytes which contain salts, we demonstrate that the presence of acetonitrile at 66% in the sample reverses the deleterious effect of salts and favors the stacking by the electrokinetic injection. The advantage of this type of stacking is that it favors certain analytes over others and it can give, in some instances, better theoretical plate numbers.

Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).

Capillary electrophoresis of double-stranded DNA in an untreated capillary.

Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.

Development of capillary electrophoresis as an alternative to high resolution agarose electrophoresis for the diagnosis of multiple sclerosis.

The presence of oligoclonal bands in cerebrospinal fluid (CSF) is used as a diagnostic indicator of multiple sclerosis (MS). These bands, gamma-globulins thought to result from a restricted antibody response directed against autoantigens or viral antigens, are consistent with CSF-specific immunoglobulin synthesis when observed in the spinal fluid and not in the serum. Current methodology commonly involves electrophoresing concentrated CSF with high-resolution agarose gel electrophoresis (HRAGE) followed by protein staining in order to visualize the oligoclonal bands. Capillary zone electrophoresis (CZE) was evaluated as an alternative method. Separation of CSF and serum proteins from 54 patients in a bare silica capillary containing a high pH borate buffer allowed for resolution of the five major zones including the gamma-region and showed a 90% concordance with the results from HRAGE banding studies. Since a simple borate buffer did not provide adequate resolution of the oligoclonal bands in the gamma-region, the separation buffer was augmented with polyethylene glycol (PEG) which provided a significant enhancement in resolution of proteins in this region (24 patient study). In addition to obtaining banding information from electropherograms obtained with these separation conditions, it was feasible to calculate a CSF Index which compared favorably with the results from nephelometry. Finally, we show that zwitterionic additives such as O-phosphorylethanolamine may further enhance resolution and that capallary electrophoresis (CE) may allow oligoclonal banding information to be obtained directly from CSF without concentration.

Analysis and classification of serum cryoglobulins.

Cryoglobulins (CG) are immunoglobulins that reversibly precipitate from serum in cold temperatures. They are classified into three types, based on the monoclonality of the γ-globulins present (1): Type I contains only monoclonal bands; type II contains mixed polyclonal immunoglobulins with a monoclonal component; and type III contains mixed polyclonal immunoglobulins only.

Myoglobin analysis.

Myoglobin represents the stores of oxygen in muscle tissues. Because of its relatively small mol wt, myoglobin is often used in electrophoretic techniques as a mol wt marker, and also as a test for separation efficiency in capillary electrophoresis (CE). The separation of myoglobin can be accomplished based on mol wt (1), based on charge, or both together (as used here).

Enzyme analysis : cathepsin d as an example.

In enzyme analysis, capillary electrophoresis (CE) offers the ease of product separation from the substrate, with the ability to use expensive reagents in microvolumes. In CE, enzymes can be measured either as mass (when they are present in high concentration) by direct light absorbency, or by catalytic activity. For example, the protease enzyme, savinase, which is used as an ingredient in washing powder, was determined directly by its absorbency at 200 nm (1). For catalytic activity measurements, the substrate, the product, or both can be measured in CE without the need for coupling reactions. Because of the increased sensitivity, most CE methods measure enzymes by their catalytic activity on a substrate. To accomplish this by CE, several approaches have been used.

Serum lamotrigine analysis.

Antiepileptic drugs vary greatly in chemical structure. Analysis of these compounds by capillary electrophoresis (CE) requires different conditions for each one, or for each group. Acidic drugs can be analyzed easily by capillary zone electrophoresis in borate buffers (1); the neutral and the mixtures can be better analyzed by micellar electrokinetic chromatography (2-4). Chapter 17 describes how phenobarbital and a few other antiepileptic drugs could be analyzed by CE.