Magnetic resonance spectroscopy in clinical chemistry: the present and the future.

Magnetic resonance spectroscopy is a powerful technique for the analysis of complex mixtures. Up to now, little of its potential in everyday clinical chemistry has been realized. An overview of the fundamentals, a discussion of the technology, and some outstanding examples of the clinical chemical research are presented. Clinical chemists are encouraged to seek out and apply this methodology to problems for which it is well suited.

Diagnosis of cancer in humans by 1H NMR of tissue biopsies.

We describe methodology for the diagnosis of human cancer, at high levels of accuracy, sensitivity, and specificity, by 1H NMR of tissue biopsies. This method is made robust and accurate by careful specimen preparation, and by multivariate analysis of spectral data. Examples are presented for the diagnosis of cancer of the prostate gland and the ovary. The potential for use of these methods noninvasively, in vivo, is shown to be very positive.

Nuclear magnetic resonance spectroscopy.

The range of problems in clinical chemistry that can be addressed by MRS is wide. The number of applications reported in the literature is growing steadily, particularly since the study of the composition of physiological fluids and tissues, and the changes thereof in disease, are well suited to study by MRS. Moreover, the major technical limitations that have impeded its progress into the clinical laboratory in the past have been addressed. Recent hardware and software developments have further improved and simplified MRS analysis. Thus, it would be surprising if MRS of physiological fluids and tissues does not become an essential technique for clinical chemists and pathologists. In practice, three main obstacles remain to be overcome: a greater availability of instruments, a larger data base of spectral changes correlated with pathological conditions, and an enhanced supply of MR-trained individuals in the clinical environment.

A rapid method for measurement of ethylene glycol.

It has been reported that ethylene glycol produces a positive interference in the triglyceride assay on the DuPont aca discrete analyzer. The sensitivity of this method for ethylene glycol was exploited to develop a rapid and convenient method for detecting and quantitating ethylene glycol in serum by the use of a "triglyceride gap." This method is based on the difference between two different enzymatic triglyceride measurements; the DuPont aca triglyceride measurement, which uses lipase and glycerol dehydrogenase, and a Boehringer Mannheim method, which uses lipase, glycerol kinase, glycerolphosphate oxidase, and peroxidase. Serum pools were spiked with increasing concentrations of ethylene glycol to construct a standard curve (linear to 25 mmol/L). Patients specimens and control sera were analyzed using a one point calibration. Within-run and between-run coefficients of variation (CV) of 1.5, 2.8, 5.8, and 3.2%; 3.4%; and 12.6% were obtained at ethylene glycol concentrations of 20.13, 8.37, and 2.18 mmol/L, respectively. The sensitivity of this method is 1.0 mmol/L. Methanol, ethanol, n-propanol, isopropanol, and acetone at 100 mmol/L; 20 mmol/L 1,3-propanediol; and 10 mmol/L glycolic acid, oxalic acid, glyoxylic acid, and lactic acid did not interfere in the quantitation of ethylene glycol. High levels (10 mmol/L) of beta-hydroxybutyrate and glycerol showed a slight positive interference on the quantitation of ethylene glycol, whereas 10 mmol/L glycoaldehyde caused a substantial overestimation of serum ethylene glycol. The presence of propylene glycol, at levels ranging from 5 to 50 mmol/L, resulted in an underestimation of serum ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotoxicosis and hungry bone syndrome--a cause of posttreatment hypocalcemia.

We report a case of a 54 year old woman with a history of recurrent Graves' disease, treated previously by thyroidectomy and later by radioiodine, who subsequently presented with tetany. Laboratory results revealed a profound hypocalcemia (total calcium 1.00 mmol/L; ionized calcium 0.53 mmol/L) and hyperphosphatemia (2.66 mmol/L) with low levels of parathyroid hormone. Although the patient's symptoms resolved after 5 days of treatment, hypocalcemia and elevated serum levels of bone-specific alkaline phosphatase (ALP, EC 3.1.3.1) activity and skeletal muscle isoenzyme (CK-MM) creatine kinase (EC 2.7.3.2) activity persisted to her discharge, 3 weeks later. Attention is drawn to the recognition and management of recalcification tetany due to the "Hungry (for calcium) Bone Syndrome," a biochemical and hormonal disturbance of calcium homeostasis and bone metabolism in the posttherapy thyrotoxic patient with hypoparathyroidism. This condition can be monitored by the use of calcium profile investigations, including bone-specific ALP, in addition to routine laboratory tests of thyroid function.

Opposite rank order of potency for alpha-2 adrenoceptor agonists on water and solute excretion in the rat: two sites and/or receptors?

Preliminary studies determined that, unlike other purported alpha-2 adrenoceptor agonists, 2,6-dimethyl clonidine (2,6-DMC) increased urine flow rate independent of vasopressin. We therefore compared the dose-response curves of three alpha-2 adrenoceptor agonists, clonidine, UK 14,304 and 2,6-DMC. Unilaterally nephrectomized Sprague-Dawley rats were anesthetized and the left kidney was exposed and the ureter cannulated. A 31-gauge needle was advanced into the renal artery to permit direct intrarenal infusion of the study drugs. All three agonists produced a dose-related increase in urine flow rate and sodium excretion. A clear opposite rank order of potency was observed when the urine flow rate was analyzed as free water and osmolar clearance. For free water clearance, clonidine much greater than UK 14,304 much greater than 2,6-DMC, with 2,6-DMC producing little change. The effect on osmolar clearance was opposite with 2,6-DMC much greater than clonidine = UK 14,304. The V2 antagonist [1-(beta-mercapto-beta,beta-pentamethylene-proprionic acid), 2-d-isoleucine,4-isoleucine]arginine-vasopressin blocked the effects of clonidine but not 2,6-DMC. In a water-loaded rat model, 2,6-DMC but not clonidine increased the delivery of filtrate out of the proximal segments of the nephron. These results are consistent with the postulate that lower doses of 2,6-DMC increase solute excretion independent of vasopressin, possibly in proximal segments of the nephron. Clonidine on the other hand increases free water clearance and this effect is mediated through an interaction with the renal actions of vasopressin. Whether these disparate effects represent two distinct receptors or two sites of alpha-2 adrenoceptors in the kidney is not known.

Potentiation of the natriuretic effect of clonidine following indomethacin in the rat.

Previous studies have demonstrated a diuretic effect of clonidine at low intrarenal infusion rates with a natriuretic effect being observed at high infusion rates (greater than or equal to 3 micrograms.kg-1.min-1). The natriuresis at high infusion rates may have been secondary to increased renal prostaglandin production. We therefore evaluated the effects of indomethacin (a cyclooxygenase inhibitor) on the response to clonidine in the anesthetized rat. Intrarenal infusions of saline (vehicle) or clonidine (0.1, 0.3, 1, and 3 micrograms.kg-1.min-1) were examined both in the presence and absence of pretreatment with indomethacin (5 mg/kg, i.p.). Clonidine produced a dose-related increase in urine volume and free water clearance at 0.3, 1, and 3 micrograms.kg-1.min-1 as compared with the vehicle group. Sodium excretion and osmolar excretion were increased only at the highest infusion rate investigated. Following indomethacin pretreatment, clonidine produced a greater increase in urine volume at each infusion rate investigated. The indomethacin pretreatment also resulted in a potentiation of the natriuretic effect of clonidine at all infusion rates. Interestingly, this was associated with an increase in osmolar clearance but not free water clearance. These effects of indomethacin were reversed by infusion of prostaglandin E2. An infusion of prostaglandin E2 attenuated the indomethacin-induced increase in both urine flow rate and sodium excretion, indicating that the effects of indomethacin were mediated by prostaglandin inhibition. These results suggest that endogenous prostaglandin production attenuates the renal effects of clonidine, and as well, that in the presence of alpha 2-adrenoceptor stimulation, prostaglandin E2 mediates an antidiuretic and antinatriuretic effect.

Role of vasopressin in response to intrarenal infusions of alpha-2 adrenoceptor agonists.

Previous studies have indicated that the effects of renal alpha-2 adrenoceptor stimulation are mediated through the blockade of the renal effects of vasopressin. If this premise is correct then 1) specific antagonists of the antidiuretic effect of vasopressin (V2 antagonists) should mimic alpha-2 adrenoceptor stimulation and 2) in the presence of V2 antagonists, the diuretic and natriuretic effect of clonidine should be attenuated. The renal effects of [d(CH2)5,D-Ile2,Ile4]AVP, a specific V2 antagonist, were studied. On the day of the experiment, uninephrectomized rats were anesthetized, and the carotid artery and jugular vein were cannulated for recording blood pressure and saline infusion, respectively. The left kidney was exposed and the ureter cannulated. A 31-gauge needle was advanced into the renal artery to permit direct i.r. infusion of study drugs. Bolus doses of the V2 antagonist (0, 1, 3, 10, or 30 nmol/kg i.v.) produced a dose-related increase in urine volume and free water clearance at all doses tested. Sodium excretion increased only at the higher doses (10 and 30 nmol/kg). This dose-related dissociation in water and then sodium excretion is similar to that observed after i.r. clonidine infusions. In the presence of the V2 antagonist, clonidine (3 micrograms/kg/min) had no effect on urine volume or free water clearance but significantly decreased the excretion of sodium from control. These results demonstrate that V2 antagonists mimic the effects of i.r. clonidine. As well, in the absence of vasopressin (V2 antagonism), the effects of clonidine are attenuated. Moreover, they are also consistent with not only an antidiuretic role for endogenous vasopressin but also an antinatriuretic one.

Enhanced natriuretic potency of intravenous clonidine: extrarenal site of action?

We have previously demonstrated that low intrarenal infusion rates of clonidine selectively increased water excretion, whereas higher infusion rates were required to increase solute excretion. This is in contrast to previous experiments where intravenous administration of clonidine resulted in a concomitant increase in water and sodium excretion. We therefore determined the dose response curve for an intravenous infusion of clonidine on water and solute excretion and compared this to the effects of an intrarenal infusion. Uninephrectomized rats were anesthetized and the remaining kidney isolated for the collection of urine. Clonidine (0.1, 0.3, 1 or 3 micrograms/kg per min) or vehicle (saline) was administered either intravenously or intrarenally. Both intravenous and intrarenal administration of clonidine produced a dose selective dissociation of water and solute excretion, that is, at low infusion rates only urine volume was increased. Higher infusion rates were required to increase sodium excretion. In addition, intravenous administration of clonidine was more potent in producing a natriuresis, suggesting that the renal effects may be, in part, secondary to additional peripheral and/or central effects of this agonist following this route of administration.

Dose selective dissociation of water and solute excretion after renal alpha-2 adrenoceptor stimulation.

In vitro studies have demonstrated an antagonism of the renal effects of vasopressin after alpha-2 adrenoceptor stimulation. Whether the effect of alpha-2 adrenoceptor stimulation in relation to sodium and water excretion in vivo is mediated through independent mechanisms is unclear. The dose-response relationship between renal alpha-2 adrenoceptor stimulation (clonidine) and water and electrolyte excretion was evaluated in anesthetized rats. Rats were nephrectomized unilaterally 7 to 10 days before the experimental day to allow isolation of renal function. A base-line level of sodium and water excretion was established by the infusion of saline (0.097 ml/min i.v.). In separate groups of rats, clonidine was infused directly into the renal artery at 0 (vehicle), 0.1, 0.3, 1 or 3 micrograms/kg/min at 0.0034 ml/min. The lower doses (0.1, 0.3 and 1 microgram/kg/min) produced a dose-related increase in urine volume and free water clearance and a decrease in urine osmolality. Electrolyte or solute excretion was not altered at these infusion rates even though urine volume increased 4-fold. The highest dose investigated (3 micrograms/kg/min) increased urine volume (9-fold) and sodium excretion (4-fold). Free water clearance and osmolar clearance were also increased. The effects of clonidine were attenuated by yohimbine but not prazosin indicating these effects were mediated by alpha-2 adrenoceptor stimulation. These results demonstrate a dose-related selectivity of alpha-2 adrenoceptor stimulation for water and sodium excretion. The increase in water excretion at the lower infusion rates would be consistent with the antagonism of the renal effects of vasopressin. The potent natriuresis observed only at higher doses indicates another mechanism may be involved.

Renal alpha 2-adrenoceptor blockade decreases sodium and water excretion in the anesthetized rat.

The reported effects of renal alpha 2-adrenoceptor blockade on sodium and water excretion have been inconsistent. We therefore studied the effect of an intrarenal infusion of an alpha 2-adrenoceptor antagonist in rats undergoing two distinct levels of diuresis and natriuresis. Renal excretion of sodium and water was studied in anesthetized rats that had been unilaterally nephrectomized (right kidney) 10 days prior to the experimental day. In the presence of the lower rate of saline infusion an intrarenal infusion of the alpha 2-adrenoceptor antagonist, yohimbine, (25.6 nmol/kg per min) resulted in no change in urine volume or sodium and potassium excretion. In the presence of the modest diuresis, due to the higher level of saline infusion, intrarenal yohimbine resulted in a decrease in urine volume, sodium excretion and free water clearance. These effects of yohimbine were not found in adrenalectomized rats. The ability to demonstrate an effect of renal alpha 2-adrenoceptor blockade was dependent on the baseline level of sodium and water excretion. These results suggest that renal alpha 2-adrenoceptors may mediate the inhibition of the renal action of vasopressin by adrenal catecholamines.

Disparate effects of neuropeptide Y and clonidine on the excretion of sodium and water in the rat.

Previous studies have demonstrated a similarity between the ability of neuropeptide Y (NPY) and clonidine to inhibit renin release and inhibit cAMP production. We therefore compared the effects of clonidine and NPY on the excretion of sodium and water in anesthetized rats which were unilaterally nephrectomized (right kidney) 10 days prior to the experiment. On the experimental day, rats were anesthetized (nembutal) and the left kidney exposed for the intrarenal infusion of the study drugs. The lowest dose of NPY (0.3 microgram/kg per min) investigated failed to alter renal function. Clonidine (0.3 microgram/kg per min) and NPY (1 microgram/kg per min) produced a similar increase in urine volume. Only NPY increased sodium excretion and osmolar clearance. Free water clearance was only increased by clonidine. Blood pressure and creatinine clearance were similar in all groups investigated. These effects were attenuated by pretreatment with pertussis toxin (5 days). The ability of pertussis toxin to block these effects suggests that the renal effects of NPY and clonidine are coupled to a G protein, conceivably the inhibitory Gi protein of the adenylate cyclase system. The disparate effects on sodium excretion and on free water and osmolar clearance indicate that the effects of these compounds may be mediated through the inhibition of different pools of hormonally stimulated cAMP.

Separan AP-273 and renal function: a novel natriuretic substance.

Recent studies have demonstrated that an anionic polyacrylamide (Separan, Union Carbide Corp.) decreased the formation of atherosclerotic lesions in the rabbit (Separan AP-30) and increased cardiac output (Separan AP-273) in the rat. Since the effect of these compounds on renal function was unknown, we investigated the dose-response relationship between Separan AP-273 and the renal excretion of electrolytes and water. In the anesthetized rat, intravenous injections of Separan at 0.01, 0.03, 0,10, and 0.30 mg/kg produced a dose-related increase in urine volume, sodium excretion, and osmolar clearance. Potassium excretion was increased less than twofold only at the maximal dose tested. At this high dose, the effect on sodium and water excretion was greater (five- and seven-fold, respectively). Creatinine clearance was not altered by these interventions. As well, the maximal dose studied was previously shown to have no effect on blood flow. These results indicate that Separan AP-273 is a potent diuretic and natriuretic substance. Taken together with similar studies using the poly(ethylene oxide) Polyox WSR N-60K, these results suggest that drag-reducing polymers may represent a novel group of compounds with diuretic and natriuretic effects.