Reagentless substrate analysis with immobilized enzymes.

By coupling an immobilized enzyme system with an electrochemical sensor, the reagent requirement for this glucose method is eliminated. Miniaturization and a further simplification of the instrumentation for the continuous analysis of glucose is achieved.

Asparaginase entrapped in red blood cells: action and survival.

As a strategy to avoid serious allergic reactions to the antitumor agent asparaginase, this enzyme was entrapped in autologous red blood cells before intravenous injection into monkeys. Additional advantages of this approach are prolonged enzyme half-life and targeting of this agent into the reticuloendothelial system.

Implanting the glucose enzyme electrode: problems, progress, and alternative solutions.

An implantable glucose sensor is needed before a reliable artificial pancreas can be realized. The principles and current status of one such device, the glucose enzyme electrode, is presented and discussed. While monitoring glucose this enzyme sensor consumes enough oxygen to become oxygen-limited. This problem has been solved by developing hydrophobic membranes that are more permeable to oxygen than to glucose. Two types of membranes with this property made from (1) cross-linked albumin and (2) sebacyl chloride (nylon) are described. Placing these membranes over the glucose enzyme electrode solves the problem of oxygen limitation. Furthermore, the addition of this type of membrane increases the linear response range of the electrode to glucose to include the entire clinical range of interest (0-400 mg/dl). Other problems in developing an implantable glucose sensor are discussed. Competing strategies to achieve an implantable artificial pancreas without using electronic or mechanical components are presented and evaluated.

Evaluation of a subcutaneous glucose sensor out to 3 months in a dog model.

OBJECTIVE: To advance the feasibility of an implantable long-term glucose sensor with bioprotective sensor membranes and test protocols using a somatostatin analog (octreotide). RESEARCH DESIGN AND METHODS: Implantable sensors were constructed with one of eight bioprotective membranes and screened in vitro for stable response to glucose. Sensors were implanted subcutaneously into nondiabetic mongrel dogs and monitored at 4-min intervals via radiotelemetry. When implanted sensor responses showed evidence of tracking blood glucose after glucagon challenge (8-21 days postimplant), a glucose infusion protocol was used to assess performance. Sensor data were collected every 4 s after octreotide inhibition of endogenous insulin release. Reference plasma glucose samples were taken every 4-10 min. RESULTS: Preimplant in vitro testing of sensors verified linearity to 33.3 mM glucose and response times to 90% of equilibrium in 2-7 min. Ten implanted sensors tracked glucose for 20-114 days, during which 25 separate glucose infusion studies were conducted. The resulting regression data yielded a mean slope of 0.99 +/- 0.06, an intercept of 0.24 +/- 0.53 mM glucose, and a correlation coefficient 0.98 +/- 0.01. Long-term sensor stability was not judged adequate for clinical application, although two sensors tracked within +/- 15% for 33 and 42 days. In vivo oxygen delivery was shown to affect sensor performance. On explant, two of eight tested bioprotective membranes were found to be biostable and to fully protect the sensor's enzyme membrane. The foreign body capsule was adequately vascularized adjacent to the sensor up to 91 days postimplant. Sensor units eventually failed because of electronic problems (package leakage) or because of biodegradation or biofouling of test bioprotective membranes. CONCLUSION: Further development of this type of sensor may provide diabetic patients with a better means of monitoring blood glucose.

A subcutaneous glucose sensor with improved longevity, dynamic range, and stability of calibration.

OBJECTIVE: To evaluate the lifetime, response time, linearity, glucose range, and calibration stability of two different types of continuous glucose sensor implants in a dog model. RESEARCH DESIGN AND METHODS: Glucose sensors based on the enzyme electrode principle that are coupled to a radio transmitter were evaluated on the bench top, sterilized, and then implanted subcutaneously in nondiabetic mongrel dogs. A multichannel radio receiver and PC data processor were used to record the sensor glucose data. Initial early reliable sensor responsivity was recognized by a vigorous hyperglycemic excursion after an intramuscular injection of glucagon. Periodically the dogs were made temporarily diabetic by blocking pancreatic insulin secretion by subcutaneous injection of a synthetic somatostatin (octreotide). By using exogenous insulin injection followed by intravenous glucose infusion, glucose levels were manipulated through the entire clinical range of interest: 2.2-38.9 mmol/l (40-700 mg/dl). Every 5-10 min, reference blood glucose samples were obtained and run in our hospital clinical laboratory. The glucose sensor data was evaluated by linear least squares optimization and by the error grid method. RESULTS: Beginning as early as postimplant day 7, the in vivo performances of sensors were evaluated by using glucose infusion studies performed every 1-4 weeks. Bench-top and in vivo 90% response-time sensors were in the range of 4-7 min during sensor lifetime. Best-performing sensors from both types are summarized as follows. The earlier-stage technology was less linear with a dynamic range of no more than 22 mmol/l glucose, had a best-case recalibration interval of 18 days, and had a maximum lifetime of 94 days. The improved later-stage technology sensors, which were constructed with the addition of bioprotective and angiogenic membranes, were linear over the full extended range of clinical interest (2.2-38.9 mmol/l [40-700 mg/dl glucose]), had a best-case recalibration interval of 20 days, and had a maximum lifetime of >160 days. CONCLUSIONS: Stable clinically useful sensor performance was demonstrated as early as 7 days after implantation and for a sensor lifetime of 3-5 months. This type of subcutaneous glucose sensor appears to be promising as a continuous and painless long-term method for monitoring blood glucose. Specifically sensors with top-layer materials that stimulate angiogenesis at the sensor/tissue interface may have better dynamic measurement range, longer lifetimes, and better calibration stability than our previously reported sensors.

Laboratory evaluation of new reusable blood glucose sensor.

An enzyme-electrode sensor designed specifically for pocket-portable self-monitoring of blood glucose is described. The sensing device in this instrument is unique because it is reusable for at least 30 days, at which time it is easily replaced by placing a new enzyme-membrane cartridge over the electrode. As little as 7 microliters of undiluted whole blood, plasma, or serum is applied directly to the sensor, and glucose is automatically determined in 30 s. No manual timing or wiping step is required after sample application. On eight production instruments, plasma glucose concentration was determined (n = 20) at 57, 125, 246, and 347 mg/dl. The average coefficient of variation for the 80 determinations for each instrument ranged from 2 to 5%, averaging 3.7%. The instrument is inherently linear, independent of hematocrit, and without oxygen limitation when dissolved oxygen concentration is greater than 35 mmHg. No interferences were found from plasma constituents, heparin, or acetaminophen.

Massive rhabdomyolysis and multiple organ dysfunction syndrome caused by leptospirosis.

We report a case of leptospiral infection in a 63-year-old man who acquired the infection while swimming in canals and streams in Hawaii. The patient's course was atypical in that he was anicteric and had no evidence of meningitis when he presented with fever, rapidly progressive and severe rhabdomyolysis, thrombocytopenia, acute renal failure, and respiratory distress syndrome. Although he recovered after a protracted illness, he required major life support, including mechanical ventilation and hemodialysis. Initial antimicrobial therapy was designed to cover major bacterial and atypical pathogens, including leptospires. An in-depth work-up for causes of this catastrophic illness confirmed acute leptospirosis. Although rare, leptospirosis is a potentially lethal infection classically associated with hepatitis, azotemia, and meningitis. Most patients experience self-limited illness, with fever, myalgias, and malaise followed by an immune-mediated aseptic meningitis. A small proportion develop shock and multiple organ dysfunction. Whereas myalgias are ubiquitous in leptospiral infection, and most patients show mildly elevated muscle enzymes, life-threatening rhabdomyolysis is rare. This atypical case is reported to urge clinicians to consider leptospirosis in the evaluation of a patient with cryptogenic sepsis who develops multiple organ dysfunction associated with rhabdomyolysis. Appropriate antimicrobial therapy, with penicillin or doxycycline, can be life-saving.

A telemetry-instrumentation system for monitoring multiple subcutaneously implanted glucose sensors.

An implantable potentiostat-radiotelemetry system for in vivo sensing of glucose is described. An enzyme electrode sensor measures the oxidation current of hydrogen peroxide formed by the stoichiometric conversion of glucose substrate and oxygen cofactor in an immobilized glucose oxidase layer. The sensor current is converted to a frequency and transmitted at programmable intervals (4, 32, 256 s) to a remote receiver. Low power CMOS circuitry is employed and device operation for up to 1.5 years is predicted using two series connected 250 mAh lithium cells. Crystal controlled RF frequencies uniquely identify each sensor allowing over 10 sensors within the same 10 m radius. A custom interface card allows a PC to program the receiver and handle the transmitted sensor data using software written in Microsoft C and QuickBasic. Software control allows on-the-fly sensor addition or subtraction to the sensor group being monitored. Over 10 sensors can be tracked long-term using the longest transmit interval, or four sensors can be tracked during short-term infusion studies when the transmit interval is reduced to 4 s. The design, construction, operation, and performance of the system hardware and software are described and evaluated.

Clinicopathologic, enzymatic, and genetic features in a case of Fabry's disease.

We report renal lesions and functional alterations in a 32-year-old man with Fabry's disease (ceramidetrihexosidase deficiency). By light microscopy of a renal biopsy specimen, distinctive "foamy" cytoplasmic alterations were observed in renal glomerular, tubular, vascular, and interstitial cells. Histochemical analysis of vacuolated epithelial cells showed glycolipid- and phospholipid-like material. Ultrastructurally, dense osmiophilic as well as stacked and concentric laminated profiles were observed within these epithelial cells. In addition, glomerular endocapillary, parietal, and vascular epithelial cells contained opaque osmiophilic granular deposits with paracrystalline arrays. Renal function studies indicated a glomerular filtration rate of 86.1 mL/min/1.73 sq m, effective renal plasma flow of 415 mL/min/1.73 sq m, tubular reabsorption of glucose of 356 mg/min/100 glomerular filtration rate, and maximal urinary concentrating and diluting ability of 568 and 46 mOsm/kg, respectively. Serum ceramide hexosidase activity was 0.18 nmole/hr/mL (normal, 8 to 15). We conclude that renal dysfunction associated with Fabry's disease is associated mainly with accumulation of glycolipid and phospholipid compounds in the walls of blood vessels and distal nephrons.

Enzymatic glucose sensors. Improved long-term performance in vitro and in vivo.

We studied the long-term in vitro and in vivo performance of enzyme electrode glucose sensors. Single commercially produced enzyme-active membranes remained functional for estimating glucose in vitro for 14-36 months. These membranes were implanted subcutaneously in rats for 1 year and, upon explanation, remained functional for measuring glucose in vitro. Sensors with these membranes plus an additional outer membrane with lower glucose permeability allowed glucose monitoring in the low oxygen tension of subcutaneous tissue. These sensors were surgically implanted in three nondiabetic dogs. Each sensor implant was coupled to a radio transmitter to allow continuous long-term glucose monitoring in these awake unrestrained dogs. In vivo sensor performance was evaluated by intravenous glucose infusion, with reference blood glucose determinations made in the clinical laboratory. These subcutaneously implanted sensors tracked changes in plasma glucose for up to 12 weeks. The in vivo initial response for three sensor implants was approximately 35 sec (n = 8). Sensor peak response to glucose after bolus infusion ranged from 3 to 14 min. Stability of sensor sensitivity within +/- 15% for more than 1 month was demonstrated in two of the dogs. Sensor lifetime was limited not by loss of enzyme activity, but by biodegradation of the outermost polyurethane membrane. The findings suggest that long-term continuous monitoring of blood glucose using a subcutaneously implanted enzyme electrode sensor may be possible.

Functional anatomy of the equine tarsocrural collateral ligaments.

Equine tarsocrural collateral ligaments (CL) were dissected grossly. The areas of attachment and fiber arrangements were described for the long lateral CL, long medial CL, 3 short lateral CL, and 3 short medial CL. Sequential cutting of CL in any order indicated that the short medial CL were responsible for the snap-joint phenomenon observed at the equine tarsocrural joint.

Anatomy of the tarsal tendons of the equine tibialis cranialis and peroneus tertius muscles.

Tendons of insertion of the equine tibialis cranialis muscle and peroneus tertius muscle (PT) were dissected grossly. Precise areas of tendon attachment and fiber arrangements within the tendons were described for the dorsal and medial tendons of the tibialis cranialis, and for the superficial lateral, deep lateral, dorsal, and medial tendons of the PT. Direct attachment of the dorsal and medial tendons of the PT into the periosteum of the central and 3rd tarsal bones and the 3rd metatarsal bone indicates that the PT may be involved in the pathogenesis of hock lamenesses.

Fascial compartments of the equine crus.

The deep fascia of the equine crus was dissected grossly and separated into 2 layers, the superficial and deep laminae of the deep fascia. Attachments of these fascial laminae to the tibia and fibula formed 5 separate osteofascial compartments: cranial, lateral, caudal deep, caudal intermediate, and caudal superficial. Cranial tibial vessels and the deep peroneal nerve entered the cranial compartment through separate fascial hiatuses; this may predispose the equine crus to the occurrence of compartmental syndromes with clinically recognizable neural deficits.

Vascular supply of the equine stifle joint.

The vascular supply of the equine stifle joint was investigated, using latex vascular injections of pelvic limbs from 3 adult horses and 6 ponies. Vessels were grossly dissected to the small arteriole level. The primary source of blood supply was the femoral artery and its branches which entered the joint on the caudal and medial surfaces. The superficial vasculature arose from the caudal branch of the deep circumflex iliac artery cranially and laterally, and from the saphenous and descending genicular arteries medially. The deep vasculature arose from the popliteal artery and its branches on the caudal joint surface. These 2 layers of vessels arborized to form the vascular rete which surrounded the joint and gave off branches to supply the articular capsule and the intraarticular structures. Venous drainage paralleled arterial pathways.