Systemic cholinergic agents induce seizures and brain damage in lithium-treated rats.

Administration of pilocarpine or physostigmine to rats treated with lithium chloride produced sustained limbic seizures, widespread brain damage, and increased concentrations of D-myo-inositol-1-phosphate (a metabolite of the phosphoinositides, lipids involved in membrane receptor function) in the brain. The syndrome was preventable with atropine. The physostigmine doses and concentrations of blood lithium that caused the syndrome are similar to those considered appropriate for psychiatric chemotherapy.

Monitoring the time course of cerebral deoxyglucose metabolism by 31P nuclear magnetic resonance spectroscopy.

The phosphorylation of 2-deoxyglucose by the mammalian brain is used as an index of the brain's energy metabolism. The results of phosphorus-31 nuclear magnetic resonance (31P NMR) monitoring of conscious animals in vivo showed rapid phosphorylation of 2-deoxyglucose by brain tissue. The rate of phosphorylation as determined by 31P NMR was consistent with results achieved by tracer methods using carbon-14-labeled 2-deoxyglucose. However, the disappearance of 2-deoxyglucose-6-phosphate was shown to be faster than that reported by tracer studies and occurred without alterations of intracellular pH and energy homeostasis. These results were confirmed by gas chromatography and mass spectroscopy. It is postulated that 2-deoxyglucose may be metabolized in several ways, including dephosphorylation by a hexose phosphatase.

Glucose-induced microvascular functional changes in nondiabetic rats are stereospecific and are prevented by an aldose reductase inhibitor.

Exposure of skin chamber granulation tissue vessels in nondiabetic rats to 11 or 15 mM D-glucose (but not L-glucose or 3-O-methylglucose) twice daily for 10 d induces vascular functional changes (increased albumin permeation and blood flow) identical to those in animals with mild or severe streptozotocin diabetes, respectively. These vascular changes are strongly linked to increased metabolism of glucose via the sorbitol pathway and are independent of nonenzymatic glycosylation as well as systemic metabolic and hormonal imbalances associated with the diabetic milieu. (J. Clin. Invest. 1990. 85:1167-1172.)

Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels.

The present experiments were undertaken to examine the hypothesis that glucose-induced increased de novo synthesis of 1,2-diacyl-sn-glycerol (which has been observed in a number of different tissues, including retinal capillary endothelial cells exposed to elevated glucose levels in vitro) and associated activation of protein kinase C may play a role in mediating glucose-induced vascular functional changes. We report here that twice daily instillation of 30 mM glucose over 10 d in a rat skin chamber granulation tissue model induces approximately a 2.7-fold increase in diacylglycerol (DAG) levels (versus tissues exposed to 5 mM glucose) in association with marked increases in vascular clearance of albumin and blood flow. The glucose-induced increase in DAG levels as well as the vascular functional changes are prevented by addition of 3 mM pyruvate. Pharmacological activation of protein kinase C with the phorbol ester TPA in the presence of 5 mM glucose increases microvascular albumin clearance and blood flow, and similar effects are observed with 1-monoolein (MOG), a pharmacological inhibitor of the catabolism of endogenous DAG. A pharmacological inhibitor of protein kinase C (staurosporine) greatly attenuates the rise in microvascular albumin clearance (but not the rise in blood flow) induced by glucose or by MOG. These findings are compatible with the hypothesis that elevated concentrations of glucose increase tissue DAG content via de novo synthesis, resulting in protein kinase C activation, and that these biochemical events are among the factors that generate the increased microvascular albumin clearance.

Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.

Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells.

Galactose ingestion increases vascular permeability and collagen solubility in normal male rats.

In view of the similarity of cataracts and neuropathy in galactose-fed and diabetic rats, the present experiments were undertaken to determine whether consumption of galactose-enriched diets (10, 25, or 50% by weight) also increases collagen crosslinking and permeation of vessels by 125I-albumin analogous to that observed in diabetic rats. The observations in these experiments: demonstrate that consumption of galactose-enriched diets for 3 wk selectively increases 125I-albumin permeation of the same vascular beds affected in diabetic rats and by diabetic vascular disease in humans (i.e., the aorta and vessels in the eye, kidney, sciatic nerve, and new tissue formed in the diabetic milieu); demonstrate that the susceptibility of the vasculature to aldose reductase-linked injury (increased permeability) varies greatly in different tissues; indicate that collagen solubility (crosslinking) changes in galactose-fed rats differ sharply from those in diabetic rats; and provide new evidence that consumption of galactose-enriched diets induces a hypogonadal state in male rats.

Effects of aldose reductase inhibitor sorbinil on neuroaxonal dystrophy and levels of myo-inositol and sorbitol in sympathetic autonomic ganglia of streptozocin-induced diabetic rats.

Biochemical and ultrastructural effects of the aldose reductase inhibitor sorbinil were examined in two experimental rat models of chronic diabetic neuropathy: rats with streptozocin-induced diabetes (STZ-D) and rats fed a galactose-enriched diet. The frequency of neuroaxonal dystrophy in the superior mesenteric sympathetic ganglia of rats with untreated 8-mo STZ-D increased sevenfold compared with that in age-matched controls. Animals chronically maintained on a diet containing 50% galactose, however, did not develop neuroaxonal dystrophy in excess of that found in untreated age-matched control rats. Institution of sorbinil therapy at the time of induction of STZ-D decreased, but did not completely normalize, the frequency of neuroaxonal dystrophy without altering the severity of diabetes; this finding is based on measurements of plasma glucose, body weight, food consumption, 24-h urine volume, and levels of glycosylated hemoglobin. Sorbitol levels in the superior cervical sympathetic ganglia (SCG) of untreated 8-mo-diabetic animals increased three- to fourfold compared with levels in controls. The increase in sorbitol content of diabetic SCG was completely prevented by early institution of dietary sorbinil therapy. The myo-inositol content of 8-mo-diabetic SCG was modestly decreased compared with controls. Sorbinil administration improved but did not completely normalize diabetic SCG myo-inositol. The sorbitol content of the SCG, superior mesenteric and celiac sympathetic ganglia, and a major trunk of the superior mesenteric nerve of short-term (2.5-mo)-diabetic rats increased comparably, but only the diabetic SCG showed a decrease in myo-inositol.

Effect of rapid normalization of plasma glucose levels on microvascular dysfunction and polyol metabolism in diabetic rats.

Effects of rapid normalization of plasma glucose levels (by insulin infused via Alzet pumps implanted intraperitoneally) on plasma insulin-like growth factor I (IGF-I) levels, granulation tissue polyol levels, and vascular permeation by 125I-labeled albumin were examined in male Sprague-Dawley rats with streptozocin-induced (60-65 mg/kg) diabetes. Two days after implantation of pumps, plasma insulin levels were twice normal levels and remained elevated (1.4-2.5 times normal) throughout the remainder of the study. Plasma glucose levels and granulation tissue polyol levels were normalized within 2 days after initiation of insulin treatment. Plasma IGF-I levels were significantly increased (2 times) by 2 days, but were not normalized until 7 days. In contrast, 125I-albumin permeation normalized at a much slower relatively linear rate and was still not completely normal after 14 days of insulin treatment. In view of 1) previous studies demonstrating that diabetes-induced increases in 125I-albumin permeation in this tissue are linked to increased metabolism of glucose to sorbitol and 2) the rapid normalization of tissue polyol levels in this study, the relatively linear rate of normalization of vascular permeability over 14 days in these studies suggests that impaired vascular barrier functional integrity in this model is mediated by structural and/or functional vascular alterations associated with sustained increased polyol metabolism rather than by increased polyol levels per se and/or by readily reversible functional and metabolic alterations associated with acute increases in polyol metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Sorbinil prevents diabetes-induced increases in vascular permeability but does not alter collagen cross-linking.

In recent studies we have demonstrated a marked increase in albumin permeation of new vessels formed by angiogenesis (in subcutaneous tissue) in the diabetic milieu. Likewise, lysyl oxidase-mediated collagen cross-linking is markedly increased in the scar tissue associated with angiogenesis. The present studies were undertaken to determine whether sorbinil, a chemical inhibitor of aldose reductase that has been shown to prevent and reverse diabetic cataracts and neuropathy, also could prevent the vascular permeability and collagen cross-linking changes in this model. Vascular permeation by 125I-BSA, collagen cross-linking, and tissue levels of sorbitol, myo-inositol, and scyllo-inositol were assessed in male Sprague-Dawley rats 3 wk after injection of streptozocin and induction of angiogenesis and collagen synthesis in polyester fabric implanted subcutaneously. Sorbinil (approximately 25 mg/kg/day) added to the diet of diabetic rats reduced the diabetes-induced increases in albumin permeation by 80%, completely prevented diabetes-induced changes in tissue levels of sorbitol and myo-inositol, and markedly reduced diabetes-induced changes in tissue levels of scyllo-inositol. In contrast, sorbinil had no effect on plasma glucose levels or collagen solubility (an index of collagen cross-linking). These observations indicate that increased vascular permeability associated with diabetes is linked to imbalances in sorbitol/inositol metabolism. These findings also indicate that diabetes-induced increases in vascular permeability and in collagen cross-linking are independent phenomena and diabetes-induced increases in vascular permeability are largely preventable by treatment with an aldose reductase inhibitor in the face of high plasma glucose levels.

Modulation of hemodynamic and vascular filtration changes in diabetic rats by dietary myo-inositol.

To assess the potential of myo-inositol-supplemented diets to prevent diabetes-induced vascular functional changes, we examined the effects of diets supplemented with 0.5, 1, or 2% myo-inositol on blood flow and vascular filtration function in nondiabetic control rats and rats with streptozocin-induced diabetes (STZ-D). After 1 mo of diabetes and dietary myo-inositol supplementation, 1) 131I-labeled bovine serum albumin (BSA) permeation of vessels was assessed in multiple tissues, 2) glomerular filtration rate (GFR) was estimated as renal plasma clearance of 57Co-labeled EDTA, 3) regional blood flows were measured with 15-microns 85Sr-labeled microspheres, and 4) endogenous albumin and IgG urinary excretion rates were quantified by radial immunodiffusion assay. In STZ-D rats, 131I-BSA tissue clearance increased significantly (2- to 4-fold) in the anterior uvea, choroid-sclera, retina, sciatic nerve, aorta, new granulation tissue, diaphragm, and kidney but was unchanged in skin, forelimb muscle, and heart. myo-Inositol-supplemented diets reduced diabetes-induced increases in 131I-BSA clearance (in a dose-dependent manner) in all tissues; however, only in new granulation tissue and diaphragm did the 2% myo-inositol diet completely normalize vascular albumin permeation. Diabetes-induced increases in GFR and in urinary albumin and IgG excretion were also substantially reduced or normalized by dietary myo-inositol supplements. Increased blood flow in anterior uvea, choroid-sclera, kidney, new granulation tissue, and skeletal muscle in STZ-D rats also was substantially reduced or normalized by the 2% myo-inositol diet. myo-Inositol had minimal if any effects on the above parameters in control rats. These observations indicate that diabetes-induced increases in regional blood flow, 131I-BSA permeation, GFR, and urinary protein excretion can be markedly reduced or normalized by consumption of myo-inositol-supplemented diets that raise plasma myo-inositol levels approximately fivefold. The failure of the 2% myo-inositol diet to normalize GFR and blood flow and albumin permeation in several tissues despite markedly elevated plasma myo-inositol levels and normal or elevated tissue myo-inositol levels indicates that if vascular functional changes in these tissues are linked to altered myo-inositol levels, they are resistant to normalization by elevation of plasma myo-inositol levels. These results suggest that other factors independent of changes in relative or absolute tissue myo-inositol levels may play an important role in the pathogenesis of diabetes-induced vascular functional changes in these tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex steroid dependency of diabetes-induced changes in polyol metabolism, vascular permeability, and collagen cross-linking.

The effects of castration on diabetes-induced increases in collagen cross-linking and vascular permeability and on polyol levels in new granulation tissue formed after induction of streptozocin (STZ) diabetes were examined in male Sprague-Dawley rats. New granulation tissue formation was induced by implanting sterile polyester fabric subcutaneously (s.c.) at the time of STZ injection 3 wk before assessment of vascular permeability and collagen cross-linking. Castration was performed 10 days before implanting the fabric. The characteristic increases in collagen cross-linking (manifested by decreased solubility in 0.5 M acetic acid) and in albumin permeation of the vasculature seen in intact diabetic rats were completely prevented by castration. Net collagen accumulation was not affected by diabetes or castration. Castration also markedly diminished diabetes-induced increases in tissue levels of sorbitol and completely prevented the decreases in tissue levels of myo-inositol and scyllo-inositol observed in intact diabetic rats, but had no effect on serum glucose levels, nonenzymatic glycosylation of plasma and granulation tissue proteins, or plasma somatomedin-C levels. The demonstration that castration prevents diabetes-induced increases in vascular permeability and collagen cross-linking as well as imbalances in tissue levels of sorbitol, myo-inositol, and scyllo-inositol in this model indicates that all of these changes are sex steroid-dependent phenomena. While the pathogenesis of these vascular permeability and collagen cross-linking changes is clearly multifactorial, these new findings: indicate that the role of sex steroids in the development of late complications of diabetes may be far more important than hitherto suspected, and suggest an explanation for the clinical observation that diabetic complications are uncommon in prepubertal diabetic subjects regardless of duration of diabetes.

Increased vascular permeability in spontaneously diabetic BB/W rats and in rats with mild versus severe streptozocin-induced diabetes. Prevention by aldose reductase inhibitors and castration.

125I-labeled albumin permeation (IAP) has been assessed in various tissues in spontaneously diabetic insulin-dependent female BB/W rats and in male Sprague-Dawley rats with severe or mild forms of streptozocin-induced diabetes (SS-D and MS-D, respectively). In BB/W diabetic rats and in rats with SS-D, indices of IAP were significantly increased in tissues and vessels predisposed to diabetic vascular disease in humans, including the eyes (anterior uvea, posterior uvea, and retina), sciatic nerve, aorta, kidney, and new vessels formed after induction of diabetes. No evidence of increased IAP was observed in heart, brain, testes, or skeletal muscle in BB/W or SS-D rats. In MS-D rats, indices of IAP were increased only in the kidney and in new vessels formed after the onset of diabetes. Marked tissue differences were observed in the effects of two structurally different aldose reductase inhibitors (sorbinil and tolrestat) and of castration on diabetes-induced increases in IAP and in tissue levels of polyols in SS-D rats. Both aldose reductase inhibitors and castration completely prevented diabetes-induced increases in IAP in new vessels and in sciatic nerve in BB/W and SS-D rats. Both aldose reductase inhibitors also markedly decreased IAP in the anterior uvea (approximately 85%), posterior uvea (approximately 65-75%), retina (approximately 65-70%), and kidney (approximately 70-100%); castration reduced IAP in the anterior uvea (approximately 55%), kidney (approximately 50%), and retina (approximately 30%) but had no effect on the posterior uvea. The diabetes-induced increases in IAP in the aorta were reduced only slightly (approximately 20%) by aldose reductase inhibitors and castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors.

This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on 1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and 2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic changes. We hypothesize that 1) increases in blood flow may reflect impaired contractile function of smooth muscle cells in resistance arterioles and 2) increases in vascular 125I-BSA permeation and urinary albumin excretion reflect impaired vascular barrier functional integrity in addition to increased hydraulic conductance secondary to microvascular hypertension associated with decreased vascular resistance.

In-vivo measurement of glucose and alanine metabolism with stable isotopic tracers.

This report describes sensitive, precise microtechniques that allow quantification of glucose and alanine metabolism in vivo using stable isotope tracers. By combined gas chromatography--mass spectrometry and selected ion monitoring--deuterium and carbon-13 enrichment in blood glucose and alanine were measured with an error of less than 2 per cent. Estimation of glucose and alanine flux in dogs by stable isotope tracer techniques was compared with simultaneous measurements made in the same animal with conventional radiotracer dilution methods. Application of the described stable isotope methods to determination of glucose and alanine turnover as well as alanine-2,3-13C incorporation into glucose in adult men confirmed the safety and validity of these techniques for human investigation.

Steady-state protriptyline levels in an outpatient population.

The authors measured steady-state protriptylive levels in 12 outpatients undergoing treatment for depression. The steady-state level of protriptyline was surprisingly high compared with levels obtained when other tricyclic antidepressants were prescribed. This finding probably accounts for the effectiveness of protriptyline at low doses and its frequent side effects.

Increased ocular blood flow and 125I-albumin permeation in galactose-fed rats: inhibition by sorbinil.

125I-Albumin permeation and blood flow (assessed with 15 micron, 85Sr-labelled microspheres) were determined in the retina, choroid, anterior uvea, and brain of male Sprague-Dawley rats fed diets containing 50% dextrin (control) or 50% galactose. Blood flow was increased in the retina, choroid, and anterior uvea but not in the brain of rats fed galactose for 3 weeks and 3 months versus controls, and was normalized by sorbinil (an inhibitor of aldose reductase) in the 3-week group. After 8 months of galactose feeding, blood flow was reduced to normal levels in the retina and was slightly below normal in the choroid; blood flow remained elevated in the anterior uvea but was significantly lower than that observed at 3 weeks and at 3 months. In rats fed galactose for 8 months, sorbinil completely normalized blood flow in the choroid, and decreased, but did not normalize, blood flow in the anterior uvea. 125I-Albumin permeation was increased in the retina, choroid, and anterior uvea of rats fed 50% galactose for 3 weeks, 3 months, and 8 months versus controls, but was unchanged in the brain. Sorbinil normalized 125I-albumin permeation in all three ocular tissues in 8-month galactose-fed rats. Polyol levels were increased significantly in all three ocular tissues of 3-week galactose-fed rats; sorbinil markedly decreased, but did not normalize, polyol levels in all three tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of brain myo-inositol levels in rats increases latency to lithium-pilocarpine seizures.

Lithium pretreatment in rats potentiates the epileptogenic effects of pilocarpine and other cholinergic agonists. In order to determine if this effect of lithium could be reversed by myo-inositol, rats were pretreated with intracerebroventricular (ICV) injections of myoinositol, artificial CSF or L-chiro-inositol. Lithium chloride, 3 meq/kg was administered intraperitoneally 20-24 h prior to the subcutaneous injection of pilocarpine, 20 or 30 mg/kg. In both experiments, myo-inositol significantly prolonged the latency to the appearance of clonic seizures and lowered the pilocarpine seizure score. myo-Inositol prevented the development of clonic seizures in 50% of the rats receiving pilocarpine, 20 mg/kg. The levels of cortical myo-inositol in rats injected with myo-inositol were approximately double those of the CSF and L-chiro-inositol groups.

Diabetes-induced increases in vascular permeability and changes in granulation tissue levels of sorbitol, myo-inositol, chiro-inositol, and scyllo-inositol are prevented by sorbinil.

In a recently developed animal model, we investigated the pathogenesis of diabetic vascular disease and demonstrated that 125I-albumin permeation is markedly increased in new "granulation tissue" vessels formed in subcutaneous tissue after the onset of diabetes. The studies described in this report were undertaken to examine the effects of an aldose reductase inhibitor on diabetes-induced increases in vascular permeability in this animal model. 125I-albumin permeation was assessed 3 weeks after the subcutaneous implantation of sterile preweighed polyester fabric (to stimulate angiogenesis) in diabetic male Sprague-Dawley rats, in controls, and in diabetic rats given sorbinil approximately 12 or approximately 25 mg/kg/d mixed in ground rat chow. Sorbinil administration prevented the diabetes-induced increase in vascular permeability by approximately 60% at the lower dose and by approximately 80% at the higher dose without affecting body weight or plasma glucose levels. Diabetes-induced changes in tissue levels of sorbitol, myo-inositol, scyllo-inositol, and chiro-inositol were also prevented by the high dose of sorbinil (data were not obtained for the lower dose). These observations are consistent with evidence linking diabetic cataracts and neuropathy to imbalances in sorbitol/inositol metabolism and support the hypothesis that diabetic vascular disease as well as neuropathy and cataracts are mediated by excess metabolism of glucose through the polyol pathway. Furthermore, these observations suggest that increased vascular permeability associated with diabetic microangiopathy in humans may be prevented by inhibitors of aldose reductase without the need to normalize blood glucose levels.

Electron beam ionization mass fragmentographic analysis of tricyclic antidepressants in human plasma.

A method for the measurement, in human plasma, of all tertiary and secondary tricyclic antidepressants prescribed in the United States is described. The method uses electron beam ionization GLC-mass spectrometry, employing a computer-controlled multiple-ion detector. This method, mass fragmentography, is used with internal standards for each drug. Plasma levels to as low as 10 ng/ml of the following drugs can be measured: amitriptyline, nortriptyline, doxepin, desmethyldoxepin, imipramine, desipramine, and protriptyline. Deuterium-labeled amitriptyline and imipramine are used as internal standards for those two drugs; for the other drugs, deuterated amitriptyline, nortriptyline, desmethyldoxepin, or desipramine is used. The method can measure up to 15 samples/hr, making it practical for large-scale studies of these drugs in patients. Spectra of each drug and examples of their analysis are given.

Synthesis of sedoheptulose from non-dialyzable, endogenous substrates in mammalian tissue extracts.

Sedoheptulose was observed to be formed at the rate of 12-120 nmol/g tissue/h in dialyzed rat and bovine tissue homogenates. The compound was identified by its gas-chromatographic retention time and by its mass spectrum. A standard of [14C]-sedoheptulose was prepared from D-[14C]fructose 6-phosphate and D-erythrose 4-phosphate for use in radioactive gas-chromatographic analysis in studies of possible precursors of the sedoheptulose. The reaction was stimulated by NAD+. Evidence is presented to show that non-dialyzable sedoheptulose 7-phosphate may be the endogenous precursor. These results show that care must be exercised when using crude enzyme preparations and extremely sensitive analytical methods so that chromatographically unresolved products formed from non-dialyzable, endogenous substrates of low molecular weight do not introduce significant error in the analysis of the expected product.