Koji Nakashima

Novel instrumentation for determination of ethanol concentrations in human perspiration by gas chromatography and a good interrelationship between ethanol concentrations in sweat and blood

Abstract Novel instrumentation for the estimation of ethanol concentration in sweat is proposed. It is composed of a sampling probe attached directly to the skin surface, a sweat rate meter for measuring the total amount of sweat secreted, a cold trap and a capillary gas chromatograph. The variation of ethanol concentration in sweat after ingestion has been measured precisely at intervals of 5–20xa0min. At the same time the ethanol concentration in blood was also determined by using a clinically authorized method. Ethanol concentrations both in sweat and blood are well related. To our knowledge, this is the first time that a clear relationship has been demonstrated during the whole elapsed period after ethanol ingestion. As the proposed method uses non-invasive sampling it may be applicable to testing for ethanol instead of the normal method using blood.

Amperometric alcohol sensor based on an immobilised bacteria cell membrane

A simple amperometric alcohol sensor was developed using a cell membrane of acetic acid bacteria, a gas-permeable membrane and an oxygen electrode. The cell membrane of Gluconobacter suboxydans IFO 12528 was adsorbed on a nitrocellulose filter and attached to the Teflon membrane of the oxygen electrode and these membranes were then covered with the gas-permeable membrane (porous Teflon). When a sample solution containing ethanol was injected into the alcohol sensor system, the sensor current decreased markedly with time and reached a steady state after approximately 3 min. A linear relationship was observed between the decrease in current and the ethanol concentration up to 25 mg l–1. The sensor was found to have a good selectivity, which was better than that of an enzyme electrode with alcohol oxidase. The response of the sensor decreased at temperatures greater than 45 °C, and good responses were obtained over a period of 15 d. The sensor was applied to the determination of serum ethanol. There was a good correlation between the results obtained by the sensor and those obtained by gas chromatography (correlation coefficient r= 0.99). The current was reproducible with a relative error of ±6.2% when the ethanol concentration of the serum was 2.3 mg ml–1. The standard deviation was 0.07 mg ml–1 in nine experiments.

Determination of seminal fructose using d-fructose dehydrogenase

Seminal plasma, the liquid component of semen, is mainly secreted from the seminal vesicles and is characterized by relatively high concentrations of fructose and extremely low concentrations of glucose [l]. Fructolysis is a main energy source for sperm. Thus, assay of seminal fructose can provide information concerning the activity of the seminal vesicles and the condition of the ejaculatory ducts [2]. Various disorders of these organs may be responsible for cases of infertility [a]. Determination of seminal fructose has been done by paper chromatography 131, gas chromatography [4], calorimetry [S] and enzymic methods 1671. We describe an enzymic method for seminal fructose using bacterial membranebound D-fructose dehydrogenase (E.C. 1.1.99.11) [8.9].

Physical stimuli and emotional stress-induced sweat secretions in the human palm and forehead

Abstract When sweating is induced by emotional or physical stimuli, the observation of the behavior of active sweat glands and measurement of the sweat rate are useful for the estimation of the degree of emotional stress on humans. When a loud sound was produced behind the subjects head, a periodical damped oscillation of sweating was observed. This sweat secretion may correlate with how a human being absorbs a big surprise stimulus. The differences of the time lag for starting to secrete sweat from sweat glands may correlate with the performance of the nervous system and/or the difference of body condition with and without stress. An intake of caffeine stimulates the central nervous system, and it rises basic, mental and thermal perspiration. The degree of physical stimuli given is amplified by the dose of caffeine, and sweating becomes 1.5–2 times greater compared to that without the dose. This study shows that sweat rate can be used as a good indicator for the state of body conditions.

Alcohol-FET sensor based on a complex cell membrane enzyme system

An alcohol -FET sensor was developed by use of a complex enzyme system in a cell membrane and an ion-sensitive field effect transistor (ISFET). The cell membrane of Gluconobacter suboxydans IFO 12528, which converts ethanol to acetic acid, was immobilized on the gate of an ISFET with calcium alginate gel coated with nitrocellulose. This ISFET (1), a reference ISFET without the cell membrane (ISFET 2) and an Ag/AgCl reference electrode were placed in 5 mM Trismalate buffer (pH 5.5, 25°C), and the differential output between ISFETS 1 and 2 was measured. The output of the sensor was stabilized by adding pyrroloquinoline quinone. The response time was ca. 10 min., and there was a linear relationship between the differential output voltage and the ethanol concentration up to 20 mg l−1. The output of the sensor was stable for 40 h below 30°C. The sensor responded to ethanol, propan- 1-ol and butan- 1-ol, but not to methanol, propan-2-ol and butan-2-ol. The sensor was used to determine blood ethanol.

A Retrospective Study of Glucose Metabolism in Mothers of Large Babies

OBJECTIVE To evaluate retrospectively the glucose metabolism of women who gave birth to large babies. RESEARCH DESIGN AND METHODS Within the first 3 days of postpartum, HbA1c of the dense erythrocytes and glycated albumin were measured. The sample group was comprised of 59 women who gave birth to heavy-for-dates infants. Fifty-five women who had average-sized infants formed the control group. Both groups had normal results on screening for gestational diabetes mellitus (GDM) by 28 weeks of gestation. The dense erythrocyte fractionation was obtained by capillary centrifugation for measurement of stable HbA1c. RESULTS Mothers of heavy-for-dates infants had a significantly higher level of HbA1c of the dense erythrocytes (mean ± SD; 5.86 ± 0.68 vs. 5.50 ± 0.70%, P = 0.015) and a higher maternal body mass index (BMI) (mean ± SD; 26.5 ±2.9 vs. 24.1 ±2.3 kg/m2, P < 0.00001) than control mothers. Differences in the occurrence of high levels of HbA1c of the dense erythrocytes and maternal BMI were statistically significant (P < 0.01, P < 0.05) between the two groups, but no difference was noted in glycated albumin. There was no statistical correlation between high levels of HbA1c of the dense erythrocytes and maternal obesity. CONCLUSIONS High HbA1c levels of the dense erythrocytes and maternal obesity are independent factors that affect fetal oversize. Heavy-for-dates infants may be a consequence of maternal hyperglycemia in late pregnancy. This maternal hyperglycemia was not detected by the routine screening test for GDM.

Glycated hemoglobin of fractionated erythrocytes, glycated albumin, and plasma fructosamine during pregnancy

OBJECTIVEnTo evaluate glucose metabolism during pregnancy, we measured plasma fructosamine, glycated albumin, and the stable glycated hemoglobin of the light and dense erythrocytes.nnnSTUDY DESIGNnThe abnormal glucose tolerance group comprised patients with gestational diabetes and those with one abnormal value on a 75 gm oral glucose tolerance test. Erythrocyte fractionation was performed by capillary centrifugation.nnnRESULTSnIn normal pregnancy glycated hemoglobin of the light erythrocytes was reduced in the second and third trimesters (3.42% +/- 0.62% [mean +/- SD] [n = 306] in the first trimester, 2.15% +/- 0.48% [n = 353] in the second, and 2.06% +/- 0.58% [n = 300] in the third), and dense erythrocytes were higher in the third trimester (first 4.59% +/- 0.46%, second 4.70% +/- 0.49%, third 5.29% +/- 0.73%). Glycated albumin and fructosamine followed a pattern similar to the light erythrocytes. The group with abnormal glucose tolerance had significantly higher levels of glycated hemoglobin of the light erythrocytes in the first and third trimesters and glycated hemoglobin of the dense erythrocytes and glycated albumin in all trimesters.nnnCONCLUSIONnThe biphasic change in nonfractionated glycated hemoglobin is the sum of the lower glycated hemoglobin of the light erythrocytes and the higher glycated hemoglobin of the dense erythrocytes in late pregnancy. The stable glycated hemoglobin of fractionated erythrocytes and the glycated albumin accurately reflect maternal glucose metabolism during pregnancy.

Immediate Elimination of Labile HbA1c With Allosteric Effectors of Hemoglobin

Our aim was to find a simple method of removing labile glycosylated hemoglobin (HbA1c) from blood samples before it is measured by cation-exchange chromatography. Labile HbA1c is formed by the binding of glucose to the NH2-terminal valine of the β-chain of HbA. We sought a more competitive binder for the same site to dissociate labile HbA1c to glucose and HbA. Inorganic phosphates were found to have a strong allosteric effect and a great ability to eliminate labile HbA1c. We developed our method with 4 mM tetrapolyphosphate in the hemolyzing solution to eliminate labile HbA1c during the automatic processing (at pH 6 and heated for 2 min at 45°C) of blood samples for HbAlc estimation. This may be useful when estimating HbA1c by the manual method.

Binding of Pyrroloquinoline Quinone to Serum Albumin

When PQQ was mixed with serum albumin at neutral pH, a spontaneous binding of PQQ to the serum protein was observed. The resulting PQQ-albumin was fairly stable for chromatographic treatments and exhibited an absorption spectrum having a peak at 336 nm. PQQ-albumin showed a coenzyme activity to a quinoprotein glucose dehydrogenase (GDH) to almost the same level as seen with authentic PQQ. PQQ was proved to be associated via e-amino group of lysyl residue of serum albumin forming a Schiff base. PQQ-chromophores isolated from PQQ-albumin by acid hydrolysis showed a marked reduced coenzyme activity for GDH. In a hydrolysate of native serum albumin, a similar chromophore fractions appeared and functioned as the prosthetic group for GDH. Growth stimulation for acetic acid bacteria was seen with such PQQ-chromophores as well as authentic PQQ. Thus serum albumin could be a temporary PQQ-carrier in mammals.

Deoxyadenosine triphosphate acting as an energy-transferring molecule in adenosine deaminase inhibited human erythrocytes

Deoxyadenosine triphosphate (dATP) is present in adenosine deaminase (ADA)-deficient or ADA-inhibited human red cells and in the red cells of the opossum Didelphis virginiana. In order to investigate the functions of dATP in the red cell, red cells were treated with 2-deoxycoformycin (dCf), a powerful inhibitor of ADA, and incubated with phosphate, deoxyadenosine and glucose. These red cells in which ATP was almost completely replaced by dATP, had the same shape, lactate production, nucleotide consumption, stability of reduced glutathione, osmotic fragility and cell deformability as red cells containing ATP. Cells merely depleted of ATP showed reduced viability. This indicates that dATP compensates well for the absence of ATP and acts as an energy-transferring molecule to maintain cell viability. These results indicate that the accumulation of dATP or the reduction of ATP is not the cause of the hemolysis observed after dCf administration.