Development and testing of a simple algorithm for a glucose clamp

The glucose clamp technique is widely used in clinical research studies to maintain a constant blood glucose (BG) level during metabolic perturbation. An algorithm is used to set the rate of an intravenous glucose infusion according to measured BG concentrations. Currently used clamp algorithms are unnecessarily complex. This paper describes the development and use of a new algorithm which is simpler to implement. The algorithm employs proportional and differential feedback control. Discrete BG sampling with a sampling period of 5 min is used. Computer simulation with a two-compartment model of glucose dynamics was used to refine the algorithm. A Monte Carlo approach was adopted to examine the effects of random variations in measured BG concentrations and the elapsed time between blood sampling and adjustment of the glucose infusion rate. A coefficient of variation in BG concentration of less than 4 per cent was predicted for a euglycemic clamp. This was confirmed when the algorithm was applied in clinical research studies. This degree of BG control compares well with other published algorithms.     

A new algorithm for haplotype-based association analysis: the Stochastic-EM algorithm

It is now widely accepted that haplotypic information can be of great interest for investigating the role of a candidate gene in the etiology of complex diseases. In the absence of family data, haplotypes cannot be deduced from genotypes, except for individuals who are homozygous at all loci or heterozygous at only one site. Statistical methodologies are therefore required for inferring haplotypes from genotypic data and testing their association with a phenotype of interest. Two maximum likelihood algorithms are often used in the context of haplotype‐based association studies, the Newton‐Raphson (NR) and the Expectation‐Maximisation (EM) algorithms. In order to circumvent the limitations of both algorithms, including convergence to local minima and saddle points, we here described how a stochastic version of the EM algorithm, referred to as SEM, could be used for testing haplotype‐phenotype association. Statistical properties of the SEM algorithm were investigated through a simulation study for a large range of practical situations, including small/large samples and rare/frequent haplotypes, and results were compared to those obtained by use of the standard NR algorithm. Our simulation study indicated that the SEM algorithm provides results similar to those of the NR algorithm, making the SEM algorithm of great interest for haplotype‐based association analysis, especially when the number of polymorphisms is quite large.     

Severity score system for progressive myelopathy: development and validation of a new clinical scale

Progressive myelopathies can be secondary to inborn errors of metabolism (IEM) such as mucopolysaccharidosis, mucolipidosis, and adrenomyeloneuropathy. The available scale, Japanese Orthopaedic Association (JOA) score, was validated only for degenerative vertebral diseases. Our objective is to propose and validate a new scale addressing progressive myelopathies and to present validating data for JOA in these diseases. A new scale, Severity Score System for Progressive Myelopathy (SSPROM), was constructed covering motor disability, sphincter dysfunction, spasticity, and sensory losses. Inter-and intra-rater reliabilities were measured. External validation was tested by applying JOA, the Expanded Disability Status Scale (EDSS), the Barthel index, and the Osame Motor Disability Score. Thirty-eight patients, 17 with adrenomyeloneuropathy, 3 with mucopolysaccharidosis I, 3 with mucopolysaccharidosis IV, 2 with mucopolysaccharidosis VI, 2 with mucolipidosis, and 11 with human T-cell lymphotropic virus type-1 (HTLV-1)-associated myelopathy participated in the study. The mean ± SD SSPROM and JOA scores were 74.6 ± 11.4 and 12.4 ± 2.3, respectively. Construct validity for SSPROM (JOA: r = 0.84, P < 0.0001; EDSS: r = −0.83, P < 0.0001; Barthel: r = 0.56, P < 0.002; Osame: r = −0.94, P < 0.0001) and reliability (intra-rater: r = 0.83, P < 0.0001; inter-rater: r = 0.94, P < 0.0001) were demonstrated. The metric properties of JOA were similar to those found in SSPROM. Several clinimetric requirements were met for both SSPROM and JOA scales. Since SSPROM has a wider range, it should be useful for follow-up studies on IEM myelopathies.     

Effect of a 2-h hyperglycemic-hyperinsulinemic glucose clamp to promote glucose storage on endurance exercise performance

Carbohydrate stores within muscle are considered essential as a fuel for prolonged endurance exercise, and regimes for enhancing such stores have proved successful in aiding performance. This study explored the effects of a hyperglycaemic–hyperinsulinemic clamp performed 18 h previously on subsequent prolonged endurance performance in cycling. Seven male subjects, accustomed to prolonged endurance cycling, performed 90 min of cycling at ~65% VO_2max followed by a 16-km time trial 18 h after a 2-h hyperglycemic–hyperinsulinemic clamp (HCC). Hyperglycemia (10 mM) with insulin infused at 300 mU/m²/min over a 2-h period resulted in a total glucose uptake of 275 g (assessed by the area under the curve) of which glucose storage accounted for about 73% (i.e. 198 g). Patterns of substrate oxidation during 90-min exercise at 65% VO_2max were not altered by HCC. Blood glucose and plasma insulin concentrations were higher during exercise after HCC compared with control (p < 0.05) while plasma NEFA was similar. Exercise performance was improved by 49 s and power output was 10–11% higher during the time trial (p < 0.05) after HCC. These data suggest that carbohydrate loading 18 h previously by means of a 2-h HCC improves cycling performance by 3.3% without any change in pattern of substrate oxidation.     

A new approach to evaluate mechanistic relationships among genotoxic phenomena: validation

In order to determine its applicability for the study of genotoxicity, a recently developed method to probe for possible mechanistic relationships among toxicological phenomena was applied to the induction of mutations in Salmonella typhimurium. Since the basis of this phenomenon is understood, this would provide a test of the applicability of the new method to DNA-based mechanisms. The results presented indicate that significant relationships are indeed found among phenomena involving damage to or modification of DNA but not between them and non-genotoxic phenomena. The present results suggest that the newly developed approach could be applied to test mechanistic hypotheses involving genotoxic phenomena.     

一种新的模糊矢量量化算法

本文通过对模糊聚类（FC）的分析和改进,提出了一种适合图像编码的模糊矢量量化的方法（FVQ）,该方法在聚类过程中未对矢量间的模糊关系进行扩张,而是构造更为合理的关系,从而在计算复杂度降低的情况下能得到更好的聚类,最后利用LBG局部寻优能力对聚类结果进一步优化,所得码书较传统LBG算法有大幅提高。     

Selectagerm: A new approach to clinical bacteriology

A method of processing clinical specimens received at the laboratory on cotton wool swabs is described. The method uses a range of selective culture media to assist in the isolation of potential pathogens from mixed cultures. Because the culture media are distributed in 2 millilitre volumes in the small squares of divided plastic petri dishes it is economically possible to use the same series for every specimen. This facilitates the rapid processing of large numbers of swabs and allows the isolation of pathogens from sites where they might otherwise go unnoticed.     

A new approach to the validation of tissue microarrays

Although tissue microarrays (TMA) have been widely used for a number of years, it is still not clear how many core biopsies should be taken to determine a reliable value for percentage positivity or how much heterogeneity in marker expression influences this number. The first aim of this study was to validate the human visual semi-quantitative scoring system for positive staining of tumour tissue with the exact values determined from computer-generated images. The second aim was to determine the minimum number of core biopsies needed to estimate percentage positivity reliably when the immunohistochemical staining pattern is heterogeneous and scored in a non-binary way. Tissue sections from ten colorectal cancer specimens were stained for carbonic anhydrase IX (CA IX). The staining patterns were digitized and 400 artificial computer-generated images were generated to test the accuracy of the human scoring system. To determine the minimal number of core biopsies needed to account for tumour heterogeneity, 50 (artificial) core biopsies per section were taken from the tumoural region of the ten digitally recorded full tissue sections. Based on the semi-quantitative scores from the 50 core biopsies per section, 2500 x n (n = 1-10 core biopsies) experimental core biopsies were then generated and scores recorded. After comparison with field-by-field analysis from the tumoural region of the whole tissue section, the number of core biopsies that need to be taken to minimize the influence of heterogeneity could be determined. In conclusion, visual scoring accurately estimated the percentage positivity and the percentage tumour present in a section, as judged by comparison with the artificial images. The exact number of core biopsies that has to be examined to determine tumour marker positivity using TMAs is affected by the degree of heterogeneity in the expression pattern of the protein, but for most purposes at least four is recommended.     

清醒大鼠胰岛素钳夹术及其糖代谢变化

为准确地评价在体组织对胰岛素 (Ins)的敏感性及探讨在胰岛素抵抗 (IR)状态下机体糖代谢变化。采用 6 ,6 D2 葡萄糖作为示踪剂 ,建立了自由状态下大鼠正常血糖 -高胰岛素钳夹术 ,并动态观察了其体内糖代谢的动态改变及血浆游离脂肪酸 (FFA)和Ins浓度随时间变化的过程。大鼠在 4 8mU (kg·min)速率Ins输注下 ,血糖稳定在正常水平而肝糖输出被抑制 ,胰岛素介导的机体葡萄糖利用率较基础状态显著增加 ,游离脂肪酸 (FFA)浓度显著下降。本钳夹术平均血糖变异系数为 5 76 %。平均GIR变异系数为 6 2 5 % ,GRd为 9 17%。重复试验GIR与GRd误差范围为 1 2 %和 1 7%。以 6 ,6 D2 葡萄糖作为示踪剂建立的自由状态下的大鼠正常血糖钳夹技术具有准确、可靠 ,无放射污染等优点 ,在外源性胰岛素 -葡萄糖代谢稳定状态下 ,机体对葡萄糖利用显著增加 ,脂肪分解显著减少。     

A new oil-gap method for internal perfusion and voltage clamp of single cardiac cells

(1.) We designed a new technique to achieve fast voltage clamp, combined with internal perfusion. The single guinea-pig cardiac cell, dissociated by collagenase treatment, was stretched across an oil-gap (30–40 m wide) from a pool of Tyrode solution to a pool of internal solution. Part of the cell membrane was disrupted in the internal solution by crushing on the cell, a tapered tip of a glass capillary. Through the open end, the intracellular medium was equilibrated with test solutions and electrical current was injected for the voltage clamp of the membrane in the Tyrode pool. (2.) The capacitive transient on stepping the membrane potential decayed with a time constant of 10–60 s, depending on the capacitive area (20–80 pF). The time course was a single exponential in 46% of the atrial cells and in 66% of the ventricular cells. In these tissues the series resistance, approximated by a ratio of the time constant andC _m, was 686±180 k (n=37) in the ventricular cells or 812±143 k (n=18) in the atrial cells. The stable seal resistance (R _seal) established in the oil-gap was around 33 M in the ventricular cells and 100 M in the atrial cells. (3.) A rapid increase in the inward current followed by a slow decay was observed on repolarization over the range negative to the potassium equilibrium potential. From the inward rectification of both peak and late currents and suppressive effects of Cs⁺ on the current, the current changes were atrributed to activation and inactivation of the inward rectifier K channel. (4.) The Na current was activated by depolarization from a holding potential of –100 mV across a threshold of about –60 mV. In normal external (145 mM Na⁺) and internal (15 mM Na⁺) solutions, peak amplitude was obtained around –25 mV. The maximum chord conductance was 6.2±1.6 mS/F in 15 ventricular cells and 3.0±0.90 mS/F in 9 atrial cells in normal Tyrode solution. The process of inactivation was fitted with a sum of two exponential functions. (5.) The reversal potential of the Na current agreed well with that predicted from the Nernst equation during perfusion of 15 and 100 mM Na⁺ internal solutions in the presence of external 140 mM Na⁺. The shift of the reversal potential was completed within 30 s of switching the internal solution. (6.) This oil-gap voltage clamp technique facilitates control of the composition of both the intra- and extra-cellular media and should prove suitable for use in studies of intracellular mechanisms controlling the membrane current of enzymatically dissociated elongated cells.     

A new technique for low-volume continuous sampling of spent dialysate: a validation study

Journal of Artificial Organs - The measure of hemodialysis (HD) adequacy recommended nowadays by most guidelines, Kt/V-urea, presents significant drawbacks. Direct dialysis quantification (DDQ)...     

A calcium clamp technique in man

Calcium infusion imply a risk for cardiovascular complications. To avoid the risks, we have developed a new method for calcium infusion in man which can keep ionized calcium concentration in whole blood (WB-Ca⁺⁺) in a steady state at a predetermined level. A solution of calcium chloride (Calcii chlor. Nord.), containing 0.117 mmol Ca⁺⁺/ml water, is infused intravenously by means of an IMED 922H infusion pump. WB-Ca⁺⁺ is determined every 5–10 min with a calcium ion-selective electrode, NOVA 2®.The infusion rate is continuously adjusted. With the same initial infusion rate, 0.31 mmol Ca⁺⁺/kg b. w./hour, hypercalcemia that is WB-Ca⁺⁺ above 1.27 mmol/1 was reached within 10 min in healthy volunteers, 8 females with a mean age of 26 years (range 19–36) and 7 males with a mean age of 29 years (range 25–34).The presettled WB-Ca⁺⁺ level, 1.45 mmol/1, was obtained within 25 min and maintained in a steady state for 155 min at 1.45 ± 0.01 mmol/1 (mean ± SE) in the females and at 1.46 ± 0.01 mmol/1 in the males. The range of the individual steady state mean WB-Ca⁺⁺ was 1.42 ± 0.02 to 1.49 ± 0.02 mmol/1.No side-effects of the infusion were noted except for a slight increase in systolic and diastolic blood pressure 60 min after start of the infusion. In conclusion, the calcium clamp technique is safe and suitable for such situations where a presettled easily controlled WB-Ca⁺⁺ level is required.