R. Freaney

Design and development of a miniaturised total chemical analysis system for on-line lactate and glucose monitoring in biological samples

A miniaturised Total chemical Analysis System (μTAS) for glucose and lactate measurement in biological samples constructed based on an integrated microdialysis sampling and detection system. The complete system incorporates a microdialysis probe for intravascular monitoring in an ex vivo mini-shunt arrangement, and a silicon micromachined stack with incorporated miniaturised flow cell/sensor array. The prototype device has been developed based on state-of-the-art membrane and printed circuit board technology. The flow-through detection system is based on a three-dimensional flow circuit incorporating silicon chips with stacked micromachined channels. An integrated biosensor array (comprising enzyme sensors specific for glucose and lactate) is placed at the base of the stack allowing the detector to be incorporated within the μTAS assembly. These glucose and lactate biosensors are prepared using photolithographic techniques, with measurement based on the detection of hydrogen peroxide at glucose oxidase and lactate oxidase modified platinum electrodes. The resulting amperometric current (at 500 mV vs, Ag/AgCl) is proportional to the concentration of analyte in the sample. All instrumentation is under computer control and the complete unit allows continuous on-line monitoring of glucose and lactate, with fast stable signals over the relevant physiological range for both analytes. The microdialysis system provides 100% sampling efficiency. Sensor performance studies undertaken include optimisation of sensitivity, linearity, operational stability, background current, storage stability and hydration time. The total system (sampling and detection) response time is of the order of 4 min, with sensor sensitivity 1-5 nA mM-1 for glucose and lactate over the range 0.1-33 and 0.05-15 mM, respectively.

Parathormone-Like Effect of Magnesium Replenishment in Steatorrhea

Abstract Five of nine patients with intestinal malabsorption had tetany associated with hypomagnesemia, hypocalcemia and absence of demonstrable bone disease. Apparent failure of parathyroid response was suggested by normal or low values for serum alkaline phosphatase and renal phosphate clearance. Intramuscular magnesium therapy abolished tetany in all. In two subjects with severe magnesium depletion there was a coincident rise in serum calcium, phosphate clearance and (in one case) alkaline phosphatase. Infusion of parathyroid extract in the magnesium-depleted state resulted in increased phosphaturia but without a rise in serum calcium. After magnesium repletion, both phosphaturic and calcemic effects were seen. The results are consistent either with failure of parathormone secretion during magnesium depletion or with a reversible resistance of bone to parathormone action.

Bone density, vitamin D status, and disordered bone remodeling in end-stage chronic liver disease.

Abstract. Hepatic osteodystrophy occurs in up to 50% of patients with chronic liver disease (CLD). The aim of this study was to determine the relative contribution of increased resorption and decreased formation to hepatic osteodystrophy by measuring biochemical markers. Twenty-seven patients with advanced CLD (14 female, 13 male) were enrolled. Bone mineral density (BMD), measured at the lumbar spine, and femoral neck, were measured by dual energy X-ray absorptiometry (DXA); bone turnover was assessed using biochemical markers of bone formation and resorption. Based on WHO criteria, osteoporosis and osteopenia were present in 41% and 18% of patients, respectively. All three markers of bone resorption (free deoxypyridinoline, pyridinoline, and hydroxyproline) were increased significantly in patients with CLD. There was a less marked change in the markers of bone formation (osteocalcin, procollagen type 1 peptide, and bone alkaline phosphatase), resulting in a negative uncoupling index in 23/27 (85%) of the patients. Only two (7%) patients had biochemical changes consistent with osteomalacia. The results suggest that increased bone resorption is the predominant cause of hepatic osteodystrophy and therapeutic strategies should be designed to suppress bone resorption, especially in preparation for liver transplantation. Bone biomarkers may be useful alternatives to bone biopsy in evaluating hepatic osteodystrophy.

Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of l-lactate

A novel amperometric biosensor for the determination of lactate was constructed by first immobilizing lactate oxidase and an osmium redox polymer ([Os(bpy)(2)(PVP)(10)Cl]Cl; abbreviated Os-polymer) on the surface of a glassy carbon electrode, followed by coating with a sol-gel film derived from methyltriethoxysilane (MTEOS). The electrooxidation current of this electrode was found to be diffusion controlled. In the presence of lactate, a clear electrocatalytic oxidation wave was observed, and lactate could be determined amperometrically at 400 mV versus Ag AgCl . The concentration range of linear response, slope of linear response and detection limit were 0.1-9 mM, 1.02 microA mM(-1), and 0.05 mM, respectively. Although L-ascorbate was electrooxidized at this potential, uric acid, paracetamol and glucose were found not to interfere.

Novel instrumentation for real-time monitoring using miniaturized flow systems with integrated biosensors

A prototype miniaturized Total Chemical Analysis System (μTAS) has been developed and applied to on-line monitoring of glucose and lactate in the core blood of anaesthetized dogs. The system consists of a highly efficient microdialysis sampling interface sited in a small-scale extracorporeal shunt circuit (‘MiniShunt’), a silicon machined microflow manifold and integrated biosensor array for glucose and lactate detection with associated computer software for analytical process control. During in-vivo testing the device allowed real-time on-screen monitoring of glucose and lactate with system response times of less than 5 min, made possible by the small dead volume of the microflow system. On-line glucose and lactate measurements were made in the basal state as well as during intravenous infusion of glucose or lactate. The prototype μTAS is currently suitable for trend monitoring but refinements are necessary before application of the system for determination of individual lactate values.

Vitamin D supplementation in the elderly: Review of safety and effectiveness of different regimes

Vitamin D deficiency is common in the elderly, especially in countries where effective sunlight or exposure to sunlight is limited. Two regimes for vitamin D supplementation—low-dose daily oral administration and intermittent high-dose administration—were examined with regard to safety and effectiveness. Eleven papers reporting studies in 449 elderly subjects were reviewed. On low-dose continuous supplementation mean concentration of 25 hydroxyvitamin D (25(OH)D) ranged from 57 to 105 nmol/L compared to 55 to 87 nmol/L following high-dose supplementation. These mean values fall within the physiological range for young adults. Hypercalcemia occurred in only 3 subjects and was associated with a predisposing cause in 2 of 3 subjects. We suggest that low dose continuous supplementation (10 to 20 μg daily) is the regime of choice but high-dose intermittent supplementation (2.5 mg six monthly) may be suitable where compliance is poor.

Prevention of hypovitaminosis D in the elderly

SummaryConflicting opinions are held on the efficacy and safety of low-dose oral vitamin D supplementation. In this study, the value of short-term and long-term low-dose vitamin D supplementation (20 μg D3 daily) is demonstrated in elderly subjects. Short-term therapy readily restored serum 25-hydroxy-vitamin D (25OHD) values to normal, and ameliorated biochemical abnormalities of calcium, phosphate, and alkaline phosphatase, consistent with healing of mild osteomalacia. Prolonged therapy (16 months) maintained serum 25OHD levels within the young adult range, and was not associated with hypercalcemia. Withdrawal of therapy was associated with a steady decline in serum 25OHD levels. Low-dose vitamin D supplementation is recommended for elderly subjects to prevent the development of hypovitaminosis D osteopathy.

Osteomalacia and osteoporosis: evaluation of a diagnostic index.

Data from a retrospective study in 41 patients is used to suggest an index of bone disease. This is designed as a means of collating available results, clarifying the significance of each in diagnosing either osteomalacia or osteoporosis, and reducing the significance of a single abnormal finding--for example, a raised alkaline phosphatase activity or low serum 25 hydroxy vitamin D, when the overall index score is low. Index scores above 35% would be diagnostic of osteomalacia; scores below 15% if associated with collapsed vertebrae suggest osteoporosis. Scores between 15% and 35% would indicate the need for a bone biopsy to discriminate between osteoporosis and osteomalacia.

In vitro optimisation of a microdialysis system with potential for on-line monitoring of lactate and glucose in biological samples.

The optimisation and evaluation of the microdialysis component of a prototype miniaturised total analysis system for application in the continuous monitoring of lactate and glucose is reported. The complete unit comprises a high efficiency microdialysis sampling system, a miniaturised microflow manifold with an integrated biosensor array, together with the hardware and software necessary for controlling the flow parameters and monitoring the sensor signals. Sampling occurs via a microdialysis shunt probe which is perfused continuously with a physiological buffered saline solution. The continuous dialysate outflow is presented to the biosensor array, resulting in the appropriate amperometric signals. Aspects of technological significance addressed here include probe membrane size, perfusate flow rate, sample flow rate, temperature change, probe sterilisation procedures, and heparin content of the physiological saline solution employed.

Ionised calcium levels in ‘Normocalcaemic’ Hyperparathyroidism

SummaryFive subjects presenting with renal stone, hypercalciuria and normal total serum calcium showed persistent elevation of ionised plasma calcium and marginal elevation of plasma parathyroid hormone concentration. Neck exploration revealed a parathyroid adenoma in each, and parathyroidectomy has been followed by reduction in both plasma ionised calcium and urine calcium. Ionised calcium measurement is an important aid in the differential diagnosis of idiopathic hypercalciuria from ‘normo-calcaemic’ hyperparathyroidism.