A patient with subcutaneous-insulin resistance treated by insulin lispro plus heparin.

Severe resistance to subcutaneous insulin but sensitivity to intravenous insulin persisted for 11 years in a 23-year-old diabetic woman. Several therapeutic trials revealed that (1) intravenous regular insulin improved her metabolic control; (2) continuous subcutaneous infusion (CSII) treatment with regular insulin or insulin lispro caused hyperglycemic period with hypoinsulinemia and hypoglycemic period with hyperinsulinemia alternately; (3) adding heparin to insulin lispro in CSII resulted in dramatic increase of serum insulin level and improvement of glycemic control; and (4) regular insulin plus heparin in CSII could not increase serum insulin level and thus the glycemic values was not improved. From these results, the patient followed the insulin lispro plus heparin protocol and obtained a better glycemic control without any adverse events. Effectiveness of this therapy may lead us to further understanding of pathophysiology of this syndrome.

Steroid analysis for medical diagnosis.

Steroid assays are important for medical diagnosis of diseases related to steroid disturbances and abuse. This article reviews the recent progress in analytical methods for steroids in the clinical laboratory. The requirements for these methods are rapid, highly sensitive, specific, direct assay of conjugated steroids, the simultaneous analysis, identification of unknown steroids, and ultra-miniaturization of the separation system.

Determination of hydrogen peroxide by micro-flow injection-chemiluminescence using a coupled flow cell reactor chemiluminometer.

A novel flow cell reactor was developed for micro-flow injection determination of hydrogen peroxide (H(2)O(2)) using horseradish peroxide (HRP)-catalysed luminol chemiluminescence. The newly developed flow cell reactor for a chemiluminometer allowed mixing of the chemiluminescent reagents in front of a photomultiplier for maximum detection of the emitted light. The rapid mixing allowed a decrease in the flow rate of the pump to 0.1-0.01 mL/min, resulting in increased sensitivity of detection of light. The flow cell reactor was made by packing HRP-immobilized gels into a flow cell (Teflon tube; 6 cm x 0.98 mm i.d.) located in the cell holder of a chemiluminometer (flow-through type). The HRP-immobilized gels were made by immobilizing HRP onto the Chitopearl gel by the periodate method. H(2)O(2) specimens (50 microL) were injected into a stream of water delivered at a flow rate of 0.1 mL/min and mixed with a luminol solution (0.56 mmol/L in Tricine buffer, pH 9.2) delivered at 0.1 mL/min in the flow cell reactor. Within-run reproducibility of the assay of H(2)O(2) was 2.4% (4.85 micromol/L; flow rate 0.1 mL/min, injection interval 10 min). The reproducibility of the H(2)O(2) assay was influenced by the flow rates and the injection intervals of the H(2)O(2) specimens. As the flow rates decreased, both the light intensity and the light duration increased. Optimal light intensity was obtained at a luminol concentration of 3-8 mmol/L, but 0.56 mmol/L was sufficient for assay of H(2)O(2) in clinical specimens. At a luminol concentration of 0.56 mmol/L, the regression equation of the standard curve for H(2)O(2) (0-9.7 micromol/L) was Y = 27.5 X(2) + 394 X + 58.9 (Y = light intensity; X = concentration of H(2)O(2)) and the detection limit of H(2)O(2) was 0.2 micromol/L. This method was used to assay glucose (2.7-16.7 mmol/L) based on a glucose oxidase (20 U/mL, pH 7.4) reaction. The standard curve for glucose was Y = 167 X(2) - 351 X + 1484 (Y = light intensity; X = glucose). The within-run reproducibility for an aqueous glucose standard (2.7 mmol/L) and a control serum (glucose, 5 mmol/L) was 4.48% (n = 5) and 5.70% (n = 9), respectively.

Total free catecholamines assay by identification of its two functional groups and micro-flow injection chemiluminescence.

We have developed a novel method of assaying total free catecholamines using sulphuric acid-derivatized beads for extracting and identifying catecholamine (CA) on the surface, and assaying the peroxide produced from CA by chemiluminescence (CL). Current assay methods for CA by electrochemical determination, fluorescence and chemiluminescence need a time-consuming separation by high-performance liquid chromatography. We eliminated this separation step by identifying the two functional groups of CA using a derivatized bead and this resulted in a highly specific CA assay. The principle is as follows: the amino group of CA was trapped by ion binding with a sulphuric acid derivative immobilized on a bead, and the diol of the CA bound to the bead was converted to peroxide with imidazole under alkaline conditions. The peroxide produced was assayed by microflow injection-horseradish peroxidase-catalysed luminol chemiluminescence. We synthesized three types of sulphuric acid-derivative immobilized beads (6.5 mm i.d.). The types of immobilized sulphuric acid derivative used were straight-chain, branched chain and benzenesulphonic, respectively. The order of the three types of beads for extracting CA was: bezenesulphonic type > branched type > straight-chain type. The optimal incubation time for generating peroxide was 30 min. The peroxide generated in the reaction solution was stable with within-run reproducibility of CV 5. 7% after incubation for 80 min. The regression equation of a standard curve for dopamine was Y = 12.8 X(2) + 476X - 373 (where Y = light intensity (RLU), X = concentration of dopamine (micromol/L)). The minimum detection limit of dopamine was 0.1 micromol/L, and the within-run reproducibility of dopamine (10.5 micromol/L) was CV 4.7% (n = 5). This method is applicable to assay of total free CA without use of HPLC.

Study of the relationship between electrophoretic mobility of the diabetic red blood cell and hemoglobin A1c by using a mini-cell electrophoresis apparatus.

A mini-cell electrophoresis system using capillary tubing (50 microm inner diameter, length ca. 10 mm and volume ca. 20 nL) was applied to measure the electrophoretic mobility of red blood cells of 89 patients with diabetes on single cell level. A significant negative correlation was observed between hemoglobin A1c and the average electrophoretic mobility, with a correlation coefficient of 0.793. By statistically processing the electrophoretic mobility of each single red blood cell, it became clear that the reduction of the average value of electrophoretic mobility was caused by the reduction of the relative frequency of the red blood cell with high mobility. The cause of the average reduction was not the shift of electrophoretic mobility of all red blood cells to the lower mobility.

Chemiluminescent detection of catecholamines by generation of hydrogen peroxide with imidazole.

A novel detection method for catecholamines using imidazole was investigated using a chemiluminescence coupled flow injection system. Imidazole catalysed decomposition of catecholamines to generate hydrogen peroxide, then the hydrogen peroxide was detected by chemiluminescence. The optimal condition for generation of hydrogen peroxide from a catecholamine was to incubate the catecholamines (53 pmol) in an imidazole solution (50 mmol/L, pH 9.0, 1.0 mL) at 60 degrees C for 30 min. Peroxide-was detected by peroxyoxalate chemiluminescence, and the rank order of the light emission intensities was as follows; dopamine (100%) >epinephrine (78%) >L-DOPA (62%) >norepinephrine (58%) >deoxyepinephrine (51%) >isoproterenol (43%) >dihydroxybenzylamine (25%). The light intensities of the reaction mixtures (corresponding to 1.06 pmol catecholamines) varied depending on the chemiluminescence (CL) detection reaction, and the rank order of the light intensity was as follows; luminol CL catalysed with horseradish peroxidase (HRP) (371%) >peroxyoxalate chemiluminescence (100%) >luminol CL catalysed with ferrycyanide (62%) >lucigenin CL (15%) >pyrogallol CL (0.8%) >purpurogallin CL (0.4%) >luminol CL (0.3%). The luminol CL reaction catalysed by HRP is recommended for the detection of peroxide in this method for catecholamines.

Effect of ACE gene on diabetic nephropathy in NIDDM patients with insulin resistance.

We investigated the influence of the angiotensin-converting enzyme (ACE) gene on the onset and/or progression of diabetic nephropathy in 62 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM; type II diabetes). Because a number of factors are believed to be involved in the onset and/or progression of diabetic nephropathy, especially in patients with NIDDM, we selected the patients with well-matched risk factors, duration of disease, glycemic control, blood pressure, and others. All patients had normal renal function and none were receiving ACE inhibitors. Patients were divided into three groups according to albumin excretion rate (AER): group A, patients with an AER less than 15 microg/min (n = 29); group B, patients with an AER between 15 and 70 microg/min (n = 19); and group C, patients with an AER greater than 70 microg/min (n = 14). The glucose disposal rate was estimated using a euglycemic hyperinsulinemic clamp. We determined the mean glucose disposal rate in 132 patients with NIDDM (6.49 mg/kg/min). Patients with a glucose disposal rate less than the mean rate were considered to have a high degree of insulin resistance (n = 36). The presence of an insertion/deletion (I/D) polymorphism of the ACE gene was determined by the polymerase chain reaction method. Among patients with a high degree of insulin resistance, diabetic nephropathy was present in 2 of 11 patients with the II genotype of the ACE gene compared with 19 of 25 patients with the ID or DD genotype (P = 0.0024). The prevalence of diabetic nephropathy was greater in patients with both significant insulin resistance and the D allele (19 of 25) than in the remaining patients (14 of 37; odds ratio, 5.20). These results suggest that the ACE gene influences the onset and/or progression of diabetic nephropathy in patients with NIDDM with significant insulin resistance.

Psychiatric manifestations accompanying interferon therapy for patients with chronic hepatitis C: an overview of cases in Japan.

Thirty case reports published in Japan that refer to psychiatric symptoms accompanying interferon (IFN) therapy were examined. These papers covered a total of 49 cases. We categorized these 49 cases into 35 cases of mood disorder, 10 of delirium and four of psychotic disorder. The key findings of our study of these cases are as follows: (i) in total, 11 patients had psychiatric past histories: five patients in the mood disorders group were susceptible to the influence of social or psychological factors; (ii) whereas the symptoms of mood disorder or delirium appeared soon after IFN was administered, the symptoms of psychotic disorders appeared later. The patients with delirium displayed many neurological abnormalities, which were reduced by suspending IFN therapy. This suggests the neurological toxicity of IFN; (iii) the outcome of most patients was good; and (iv) we suspect that IFN-induced psychiatric symptoms other than delirium are connected with psychoneuroimmunological functions.

Effect of long-term 'corn peptide' ingestion on alcohol metabolism in stroke-prone spontaneously hypertensive rats with alcohol loading.

The effect of long-term 'corn peptide (CP)' ingestion on alcohol metabolism was investigated in stroke-prone spontaneously hypertensive rats (SHR-SP) with alcohol loading. Long-term CP ingestion in the EtOH/CP group did not significantly increase plasma GOT and GPT activities but markedly increased hepatic ADH and ALDH activities. Intragastric CP administration prior to a dose of 1.0 g/kg ethanol significantly lowered the blood ethanol concentration in SHR-SP which had been loaded with ethanol for a long time. Compared with non-loaded SHR-SP (control group), the rats loaded for a long time with ethanol (EtOH group) showed high concentrations of taurine, glycine and histidine in the plasma. The plasma threonine and proline concentrations were significantly elevated by long-term CP ingestion (EtOH/CP group), but the plasma alanine concentration was rather decreased. These results suggest that short- or long-term CP ingestion may enhance the alcohol metabolism within the body because of an increase in ADH and ALDH activities as well as the alleviation of alcohol-related hepatic injury.

Amines for detection of dopamine by generation of hydrogen peroxide and peroxyoxalate chemiluminescence.

A range of nitrogen-containing compounds (alkyl amines, piperazines, cyclohexylamines and nitrogen heterocyclics) were investigated for generation of hydrogen peroxide from dopamine and detection by peroxyoxalate chemiluminescence. Imidazole, ethyleneurea and allantoin among the nitrogen heterocyclic compounds tested generated hydrogen peroxide from dopamine following incubation at 60 degrees C, pH 9.5-10.5, for 0-30 min. Imidazole was the most effective for generation of hydrogen peroxide, but imidazole derivatives with a primary amine side chain (histamine) or thiol (ethylenethiourea) were not effective. The presence of a ketone group (ethyleneurea, allantoin) did not hinder the reaction. Under optimal conditions (30 min incubation, 50 mmol/L imidazole) 10.5 nmol of dopamine could be detected. The cyclohexylamines tested produced low amounts of hydrogen peroxide (0.09-2.74% of light intensity with imidazole), and the piperazines and the alkyl amines tested produced no detectable hydrogen peroxide. Imidazole reacts with the phenolic groups of dopamine in a different manner from monoamine oxidase, and a reagent containing imidazole, ethyleneurea or allantoin was useful for non-enzymatic detection of dopamine by peroxyoxalate chemiluminescence.

Detection of an amino acid polymorphism in hormone-sensitive lipase in Japanese subjects.

Hormone-sensitive lipase (HSL) plays an important role in energy metabolism by controlling the hydrolysis of triglycerides stored in adipose tissue. To investigate whether mutations in the HSL gene are associated with non-insulin-dependent diabetes mellitus (NIDDM), we screened for mutations of this gene using single-stranded conformation polymorphism (SSCP) in 35 Japanese subjects with NIDDM. SSCP analysis identified a variant pattern in axon 4, and the sequence showed that this variant pattern resulted from amino acid polymorphism (Arg309Cys). Subsequent study showed that this polymorphism was found in 18 of 151 NIDDM patients and 10 of 97 nondiabetic subjects, but allele frequency was not significantly different between the two groups (P = .7). Body mass index, serum triglyceride, and high-density lipoprotein (HDL) cholesterol were not different in subjects with and without the polymorphism. But serum total cholesterol was higher in subjects with the polymorphism than in subjects without it (P = .0005). These data indicate that this HSL polymorphism is not associated with NIDDM, obesity, and serum triglyceride level. However, an effect of the polymorphism to elevate serum total cholesterol has not been excluded, although further study is necessary to resolve its association with cholesterol metabolism.

Preparation of corn peptide from corn gluten meal and its administration effect on alcohol metabolism in stroke-prone spontaneously hypertensive rats.

Corn peptide (CP) was prepared from corn gluten meal by proteolysis with alkaline protease from alkalophilic Bacillus A-7. Free amino acids were not found in the CP product. Gel filtration on a Shodex OH-packed column revealed that the molecular weight distribution of the CP was less than about 2,000, characteristic of dipeptides to decapeptides, i.e. oligopeptides. The amino acid pattern of CP was similar to that of corn gluten meal, which was rich in alanine and branched-chain amino acids, but poor in basic amino acids. The effect of the CP administration on alcohol metabolism was examined with SHR-SP, which were given ethanol orally through a gastric tube at the rate of 1.0 g/kg. Prior administration of CP at 1.0 g/kg resulted in fast disappearance of ethanol and its oxidative product acetaldehyde from the blood relative to the control without administration. Hence, it is suggested that CP, rather than its constituent amino acids such as alanine and proline, effectively takes part in enhancing the metabolism of ethanol as well as acetaldehyde.

Detection of substances with alcoholic or phenolic hydroxyl groups by generation of hydrogen peroxide with imidazole and peroxyoxalate chemiluminescence.

On-line detection of substances with an alcoholic or phenolic hydroxyl group using imidazole and peroxyoxalate chemiluminescence was investigated qualitatively using a flow-injection method. The substances tested included six polyphenols, five monophenols and six sugars. After incubation at 80 degrees C with an imidazole buffer (pH 9.5) the substances were detected by peroxyoxalate chemiluminescence. The polyphenols tested (e.g., pyrogallol, purpurogallin, and dopamine) showed the strongest light emission. The sugars with hydroxyl groups (e.g., fructose and lactose) and the monophenols (e.g., phenol, serotonin, and beta-estradiol) produced only a weak light emission. Imidazole served two roles, it catalysed the reaction with the hydroxyl compound and initiated peroxyoxalate chemiluminescence on-line. A novel reactor formed by packing glass beads into a flow cell (Teflon) of a chemiluminometer improved the sensitivity of light detection.

The effect of PGI2 analogue on vascular endothelial function and platelet aggregation in patients with NIDDM.

The effect of the short-term administration of beraprost sodium, an analogue of prostaglandin I2 (PGI2), on the function of vascular endothelial cells and platelet in non-insulin-dependent diabetes mellitus (NIDDM) patients was investigated. Seven nonobese NIDDM patients with microalbuminuria were recruited for this study. They received a dose of 20 micrograms of beraprost sodium three times daily for 1 month. Before and after this treatment, various factors concerning functions of vascular endothelial cells and platelet were measured. Treatment with PGI2 analogue caused a decrease in basal levels of plasma lipoprotein (a) from 16.8 +/- 5.3 to 13.2 +/- 4.4 mg/dL (p < 0.05), immunoreactive-(i)endothelin from 2.4 +/- 0.3 to 1.6 +/- 0.2 pg/mL, and i-thrombomudulin from 9.3 +/- 3.7 to 7.9 +/- 3.0 FU/L, respectively, and caused a significant increase in basal plasma i-tissue type plasminogen activator (tPA) from 5.3 +/- 0.7 to 8.3 +/- 1.5 ng/mL (p < 0.01). This treatment also increased maximum response of i-tPA induced by desmopressin infusion. Platelet aggregation due to ADP was inhibited in five of six patients after this treatment. In conclusion, treatment with PGI2 analogue caused a decrease in the presumed promoting factors of angiopathy such as lipoprotein (a) and endothelin and an increase in the protecting endothelial factor of angiopathy, tissue type plasminogen activator in patients with NIDDM. And immunoreactive thrombomodulin levels which reflect the vascular endothelial cell injury tended to decrease with the treatment. Therefore, it is suggested that this treatment preserves the vascular endothelial function in diabetes.

Endothelium-dependent relaxation in peripheral vasculature and kidney of non-insulin-dependent diabetes mellitus.

Desmopressin (DDAVP), an AVP.V2-receptor agonist, evokes endothelium-dependent relaxation (EDR) due to nitric oxide (NO), EDR factor (EDRF) in the systemic vasculature, and glomerular afferent arterioles via AVP receptor(s). Glyceryl trinitrate (GTN) causes endothelium-independent (nonreceptor-mediated) vasodilation. We elucidated the possible involvement of EDRF in early non-insulin-dependent diabetes mellitus (NIDDM) and glomerular hyperfiltration (GHF) by DDAVP and GTN infusions. Patients with advanced DM nephropathy (DM.Np) (n = 7) were also examined. DDAVP and GTN decreased the mean blood pressure in DM with GHF (DM + GHF) and without GHF (DM-GHF) greater than that in normal subjects (N), without any difference in the heart rate changes in any group. Plasma levels of cGMP, a cellular messenger of NO, were significantly increased by DDAVP and GTN with a similar increment in each group. DDAVP caused a significant increase in urinary cGMP excretion in each group with a similar increment in each group. However, it caused a transient increase in creatinine clearance only in DM + GHF although GTN did not, and an exaggerated excretion of urinary albumin in early NIDDM, especially in DM+GHF, without a change in beta 2-microglobulin excretion. In contrast, in DM.Np GTN caused a decrease in blood pressure and an increase in plasma cGMP levels, but DDAVP did not. In conclusion, in peripheral vasculature and kidney, an enhanced sensitivity of vascular smooth muscle to NO is present in early NIDDM. The exaggerated dilation of glomerular afferent arterioles by preferentially produced NO in in situ, which causes a rise in PGC, might be partly responsible for the glomerular hyperfiltration and subsequently the increase in the glomerular protein permeation of DM+GHF. However, in peripheral blood vessels of DM.Np EDR is impaired. Thus, EDR seems to change with the development of NIDDM.

New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin.

The effects of various boronate compounds, 4-biphenylboronic acid, 4-bromobenzene-boronic acid, trans-4-(3-propionic acid)phenylboronic acid and 4-iodophenylboronic acid, on the horseradish peroxidase (HRP) catalysed chemiluminescent oxidation of pyrogallol and purpurogallin by peroxide were investigated. trans-4-(3-Propionic acid)phenylboronic acid produced a 13.7-fold enhancement in the peak light emission from the chemiluminescent HRP catalysed pyrogallol reaction (detection limit for HRP < 1.25 fmol). At low enhancer concentration a single peak of light emission was observed and as the enhancer concentration increased the time to peak light emission became progressively longer. The chemiluminescence showed two peaks at higher concentrations (> 54.3 mumol/L) and the individual peak times depended upon the concentration of the enhancer. All of the boronates enhanced peak light emission in the chemiluminescent HRP catalysed purpurogallin reaction. 4-Biphenylboronic acid was the most effective and it enhanced peak light emission 314-fold. The practical detection limit for HRP (Type VIA) using this enhancer was 4.18 pmol (peak emission at 20 minutes). This compound also enhanced peak light emission 232-fold from a chemiluminescent HRP-purpurogallin reaction in which molecular oxygen replaced peroxide as the oxidant.

Immunohistochemical study of human advanced glycosylation end-products (AGE) in chronic renal failure.

In patients with diabetic renal failure plasma advanced glycosylation end-products (AGE) levels are reported to be elevated and dialyzer of continuous ambulatory peritoneal dialysis (CAPD) is usually used with a high glucose concentration. Here, an immunohistochemical study on human AGE accumulation in vascular beds and peritonea of patients with chronic renal failure (CRF) or those on CAPD was undertaken. Further, the influence of aging was studied using AGE-specific monoclonal antibody. 1. AGE accumulation was observed in radial arterial walls (from vascular intima to smooth muscle layer) of diabetic patients with CRF. Even in some non-diabetic patients with CRF (n = 3/6), especially in those with a long history of CRF and dialysis treatment, similar positive staining was seen in vascular walls. No AGE staining was observed in any renal tissue of age-matched control subjects including tissue from patients with acute renal failure. 2. Although AGE accumulation was not seen in the peritonea of CRF patients with no prior CAPD therapy, it was seen in the mesothelial layers and in adjacent coarse connective tissues of peritonea from patients on CAPD (n = 6), even from as early as only 3 months of CAPD therapy. 3. AGE accumulation was observed in the vascular bed of the non-diabetic aged kidney with normal function, but not in that of the young kidney. Thus, AGE accumulation in the vascular bed may depend on the degree and term of renal impairment and on aging in addition to diabetes. AGE accumulation in the peritonea became positive following CAPD treatment, indicating that it might affect the efficiency of CAPD.

Imaging of chemiluminescent reactions in mesoscale silicon-glass microstructures.

Chemiluminescent reactions in mesoscale analytical structures (chips) containing micrometre-sized interconnecting channels and chambers (pL-nL total volume) were imaged. The chips were fabricated by bonding Pyrex glass to etched pieces of silicon using a high-temperature diffusive bonding technique. In initial experiments light emission from an enhanced chemiluminescent horseradish peroxidase reaction and from a peroxyoxalate reaction contained in straight channels (300 microns wide x 20 mu deep; volume 70.2 nL) and open chambers (812 microns wide, 400 microns deep, 5.2 mm long) linked by channels (100 microns wide, 20 microns deep) to an exit and entry port were studied using a specially modified microplate holder and an Amerlite microplate luminometer. Light emission from more complex structures (two chambers interconnected by a branching channel 100 microns wide, 20 microns deep) filled with a solution containing alkaline phosphatase, Emerald, and CSPD was imaged using a Photometrics Star 1 CCD camera. Detailed investigation of the detection and spatial resolution of the signal was performed on a Berthold Luminograph LB 980 using both the enhanced chemiluminescent horseradish peroxidase reaction and a peroxyoxalate reaction. We successfully resolved light emission from silicon structures with dimensions 100 microns wide and 20 microns deep. These simple silicon structures served as models for more complex designs that will be used for simultaneous multi-analyte assays in which an imaging system resolves and quantitates light emission from different locations on a silicon-glass analytical device.

Applications of a microfabricated device for evaluating sperm function.

Mesoscale structures (microns dimensions, nL-pL volumes) have been designed and fabricated in silicon for use in various analytical tasks. We studied sperm motility and performed sperm selection in channels (80 microns wide x 20 microns deep), branching structures (40 microns wide x 20 microns deep, eight bifurcations), and channels containing barriers (7 microns feature size). Sperm-cervical mucus and sperm-hyaluronic acid interactions were assessed by using appropriate microchannel-chamber structures filled with either cervical mucus or hyaluronic acid. Simultaneous assessment of the potency of different spermicides (e.g., nonoxynol-9, C13G) and spermicide concentrations was achieved with structures comprising chambers containing spermicide connected via channels to a central chamber into which semen was introduced. Semen was also tested for the presence of sperm-specific antibodies by using microchannels filled with human anti-IgG antibody-coated microbeads.