Alpha-thiolamines such as cysteine and cysteamine act as effective transglycating agents due to formation of irreversible thiazolidine derivatives.

Non-enzymatic glycation of proteins and some phospholipids is considered to be an important factor in the genesis of diabetic complications. While this process has been viewed traditionally as entirely non-enzymatic and unidirectional, the discovery of fructosamine-3-phosphate (FN3K) and identification of FN3K-mediated deglycation mechanisms have made it apparent that non-enzymatic glycation is not unidirectional and that it can be reversed by deglycation reactions. While FN3K operates on ketosamines, the second intermediate in the non-enzymatic glycation cascade, we recently identified another potential deglycation mechanism that can operate on Schiff bases, the first intermediates of the non-enzymatic glycation process. The initial step in this postulated deglycation process is a transglycation reaction between a L.M.W. intracellular nucleophiles and a macromolecule-bound aldosamines, which regenerate unmodified proteins or phospholipids with a concomitant production of aldose-nucleophile transglycation byproducts. In vitro, transglycation occurs readily with amino acids, polyamines, thiols and thiolamines. There are indications that this reaction also occurs in vivo since in an initial GC/MS analysis of human urine we detected significant amounts of a transglycation product, glucose-cysteine (G-Cys), which was markedly increased in diabetics. Despite these encouraging early data, it is not yet clear to what extent transglycation is important in vivo and which intracellular nucleophiles are most relevant to this process. As discussed by us previously in this journal, one likely candidate for this role is glutathione since it is distributed universally and since there are well described mechanisms for removal of S-linked glutathione adducts from cells by the multi-drug-resistance (MDR) pumps. In this paper we report on another class of likely transglycating agents, alpha-thiolamines such as cysteine and cysteamine. While concentrations of these compounds in tissues are significantly lower than those of GSH, they react with Schiff bases more rapidly than GSH and, most significantly they form stable and irreversible thiazolidine products such as glucose-cysteine (G-Cys) and glucose-cysteamine (G-Ctm) that can subsequently be removed from cells. The possibility that alpha-thiolamines may play a physiological role as deglycating agents in vivo is very attractive since it suggests a possible strategy for inhibiting nonenzymatic glycation and diabetic complications that could be readily implemented through nutritional or pharmacological approaches. Such intervention is eminently feasible since there are at least three thiolamines already approved for human use. These include cysteamine used for the treatment of cystinosis; N-acetylcysteine utilized as a mucolytic and antioxidant agent, in the therapy of acetaminophen poisoning and radiocontrast-induced nephrotoxicity; and penicillamine used for treatment of Wilson's disease. Consequently, determining whether these compounds have the expected anti-glycating effects in vivo should be relatively straightforward.

Alpha-oxoaldehyde metabolism and diabetic complications.

The factors responsible for variable susceptibility to diabetic nephropathy are not clear. According to the non-enzymatic glycation hypothesis, diabetes-related tissue damage occurs due to a complex mixture of toxic products, including alpha-oxoaldehydes, which are inherently toxic as well as serving as precursors for advanced glycation end-products. Protective mechanisms exist to control this unavoidable glycation, and these are determined by genetic or environmental factors that can regulate the concentrations of the reactive sugars or end-products. In diabetes these protective mechanisms become more important, since glycation stress increases, and less efficient defence systems against this stress could lead to diabetic complications. Some of these enzymatic control mechanisms, including those that regulate alpha-oxoaldehydes, have been identified. We have observed significant increases in production of the alpha-oxoaldehydes methylglyoxal and 3-deoxyglucosone in three human populations with biopsy-proven progression of nephropathy. The increase in methylglyoxal could be secondary to defects in downstream glycolytic enzymes (such as glyceraldehyde-3-phosphate dehydrogenase) that regulate its production, or in detoxification mechanisms such as glyoxalase. Other mechanisms, however, appear to be responsible for the observed increase in 3-deoxyglucosone levels. We present results of our studies on the mechanisms responsible for variable production of alpha-oxoaldehydes by measuring the activity and characteristics of these enzymes in cells from complication-prone and -resistant diabetic patients. New therapeutic interventions designed to control these endogenous mechanisms could potentially enhance protection against excessive glycation and prevent or reverse complications of long-term diabetes.

Enzymatic deglycation--a new paradigm or an epiphenomenon?

Following the discovery of FN3K (fructosamine 3-kinase), and more recently of FN3KRP (FN3K-related protein), research in our laboratory has been focused on testing the enzymatic deglycation hypothesis and investigating the roles of FN3K and FN3KRP. Thus far, using human erythrocytes as a model system, we have obtained the following evidence of enzymatic deglycation: (a) production of GHb (glycated haemoglobin) by D-glucose in intact erythrocytes is 5-fold lower than in haemolysates; (b) glycation of GHb by D-glucose in intact erythrocytes is 5-fold lower than that by L-glucose; (c) inhibition of ATP production in erythrocytes leads to an acceleration in the rate of GHb production by D-glucose; and (d) inhibition of FN3K in erythrocytes by a competitive inhibitor increases the accumulation of GHb. In spite of these data supporting the enzymatic deglycation hypothesis, some outstanding issues remain. These include, among others, the fact that while the apparent deglycation mechanism does not operate on L-glucose, semi-purified FN3K appears to be able to use both D- and L-fructosamines as substrates. Moreover, analysis of the fructoselysine 3-phosphate content of haemoglobin from diabetic subjects suggests that, in addition to FN3K, another deglycating mechanism may be operative in human erythrocytes. Elucidation of these issues is a challenge in the evolving field of deglycation research. Most important, however, is the question of whether enzymatic deglycation is truly an important defence mechanism or merely an epiphenomenon.

Human fructosamine-3-kinase: purification, sequencing, substrate specificity, and evidence of activity in vivo.

Nonenzymatic glycation appears to be an important factor in the pathogenesis of diabetic complications. Key early intermediates in this process are fructosamines, such as protein-bound fructoselysines. In this report, we describe the purification and characterization of a mammalian fructosamine-3-kinase (FN3K), which phosphorylates fructoselysine (FL) residues on glycated proteins, to FL-3-phosphate (FL3P). This phosphorylation destabilizes the FL adduct and leads to its spontaneous decomposition, thereby reversing the nonenzymatic glycation process at an early stage. FN3K was purified to homogeneity from human erythrocytes and sequenced by means of electrospray tandem mass spectrometry. The protein thus identified is a 35-kDa monomer that appears to be expressed in all mammalian tissues. It has no significant homology to other known proteins and appears to be encoded by genomic sequences located on human chromosomes 1 and 17. The lability of FL3P, the high affinity of FN3K for FL, and the wide distribution of FN3K suggest that the function of this enzyme is deglycation of nonenzymatically glycated proteins. Because the condensation of glucose and lysine residues is an ubiquitous and unavoidable process in homeothermic organisms, a deglycation system mediated by FN3K may be an important factor in protecting cells from the deleterious effects of nonenzymatic glycation. Our sequence data of FN3K are in excellent agreement with a recent report on this enzyme by Delpierre et al. (Diabetes 49:1627-1634, 2000).

Glycated proteins in diabetes.

The term nonenzymatic glycation (of protein) refers to a wide variety of spontaneous reactions between reducing sugars and protein-bound amines. This reaction has been documented in humans and plays a role in the development of diabetic complications and perhaps in some of the degenerative processes of aging. In addition to the monocarbonyl sugars and their derivatives, an additional source of glycation is alpha-dicarbonyls. Over time, nonenzymatic glycation leads to the formation of irreversible terminal products known collectively as advanced glycation end-products (AGE) and extensive data on the role of AGEs in the etiology of diabetic complications exist. Our improved ability to measure alpha-dicarbonyls and specific AGEs may provide new and more powerful tools to monitor diabetes and predict diabetic complications in the future.

alpha-Dicarbonyls increase in the postprandial period and reflect the degree of hyperglycemia.

OBJECTIVE: Chronic hyperglycemia is known to increase tissue glycation and diabetic complications, but controversy exists regarding the independent role of increased postprandial glucose excursions. To address this question, we have studied the effect of postprandial glycemic excursions (PPGEs) on levels of methylglyoxal (MG) and 3-deoxyglucosone (3-DG), two highly reactive precursors of advanced glycation end products (AGEs). RESEARCH DESIGN AND METHODS: We performed 4-month crossover studies on 21 subjects with type 1 diabetes and compared the effect of premeal insulin lispro or regular insulin on PPGEs and MG/3-DG excursions. PPGE was determined after standard test meal (STMs) and by frequent postprandial glucose monitoring. HbA1c and postprandial MG and D-lactate were measured by HPLC, whereas 3-DG was determined by gas chromatography/mass spectroscopy. RESULTS: Treatment with insulin lispro resulted in a highly significant reduction in PPGEs relative to the regular insulin-treated group (P = 0.0005). However, HbA1c levels were similar in the two groups, and no relationship was observed between HbA1c and PPGE (P = 0.93). Significant postprandial increases in MG, 3-DG, and D-lactate occurred after the STM. Excursions of MG and 3-DG were highly correlated with levels of PPGE (R = 0.55, P = 0.0002 and R = 0.61, P = 0.0004; respectively), whereas a significant inverse relationship was seen between PPGE and D-lactate excursions (R = 0.40, P = 0.01). Conversely, no correlation was observed between HbAlc and postprandial MG, 3-DG, or D-lactate levels. CONCLUSIONS: Increased production of MG and 3-DG occur with greater PPGE, whereas HbA1c does not reflect these differences. Reduced PPGE also leads to increased production of D-lactate, indicating a role for increased detoxification in reducing MG levels. The higher postprandial levels of MG and 3-DG observed with greater PPGE may provide a partial explanation for the adverse effects of glycemic lability and support the value of agents that reduce glucose excursions.

Metformin reduces systemic methylglyoxal levels in type 2 diabetes.

Methylglyoxal (MG) is a reactive alpha-dicarbonyl that is thought to contribute to diabetic complications either as a direct toxin or as a precursor for advanced glycation end products. It is produced primarily from triose phosphates and is detoxified to D-lactate (DL) by the glyoxalase pathway. Because guanidino compounds can block dicarbonyl groups, we have investigated the effects of the diamino biguanide compound metformin and of hyperglycemia on MG and its detoxification products in type 2 diabetes. MG and DL were measured by high-performance liquid chromatography in plasma from 57 subjects with type 2 diabetes. Of these subjects, 27 were treated with diet, sulfonylureas, or insulin (nonmetformin), and 30 were treated with metformin; 28 normal control subjects were also studied. Glycemic control was determined by HbA1c. MG was significantly elevated in diabetic subjects versus the normal control subjects (189.3 +/- 38.7 vs. 123.0 +/- 37 nmol/l, P = 0.0001). MG levels were significantly reduced by high-dosage (1,500-2,500 mg/day) metformin (158.4 +/- 44.2 nmol/l) compared with nonmetformin (189.3 +/- 38.7 nmol/l, P = 0.03) or low-dosage (< or = 1,000 mg/day) metformin (210.98 +/- 51.0 nmol/l, P = 0.001), even though the groups had similar glycemic control. Conversely, DL levels were significantly elevated in both the low- and high-dosage metformin groups relative to the nonmetformin group (13.8 +/- 7.7 and 13.4 +/- 4.6 vs. 10.4 +/- 3.9 micromol/l, P = 0.03 and 0.06, respectively). MG correlated with rising HbA1c levels (R = 0.4, P = 0.03, slope = 13.2) in the nonmetformin subjects but showed no increase with worsening glycemic control in the high-dosage metformin group (R = 0.0004, P = 0.99, slope = 0.02). In conclusion, MG is elevated in diabetes and relates to glycemic control. Metformin reduces MG in a dose-dependent fashion and minimizes the effect of worsening glycemic control on MG levels. To the extent that elevated MG levels lead to their development, metformin treatment may protect against diabetic complications by mechanisms independent of its antihyperglycemic effect.

Study of the metabolism of choline and phosphatidylcholine in tumors in vivo using phosphonium-choline.

The results of an initial study on the feasibility of using the phosphonium analog of choline to follow the metabolism of phosphatidylcholine in tumors in vivo using 31P NMR are reported. C3H/He mice bearing a mammary carcinoma tumor on the foot pad were fed a choline-free diet supplemented with the phosphonium analog of choline. Metabolites of this compound, including the phosphonium analogs of phosphatidylcholine, phosphocholine, glycerophosphocholine, and betaine were observed noninvasively in vivo in tumors by 31P NMR after 2-3 weeks of feeding. Clearance of these phosphonium-labeled metabolites from tumors was measured after a change to a choline-containing diet. Significant decreases were seen in the levels of the analogs of betaine (P < 0.003) and phosphatidylcholine (P < 0.004) by Day 4. A significant increase in the level of authentic phosphocholine (P < 0.003) occurred over the same time period.

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts.

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

Quantitation of 3-deoxyglucosone levels in human plasma.

3-Deoxyglucosone (3DG), a reactive dicarbonyl, is an important intermediate in the formation of advanced glycation end products (AGEs). The AGEs are particularly important in diabetes since they have been correlated with the development of diabetic complications. Consequently, measurements of 3DG are likely to provide valuable insights into the role of this metabolite in the etiology of diabetic complications. While several methods of 3DG quantitation in human plasma have been previously published, a significant discrepancy (over 30-fold) exists in the reported values. Knecht et al. (Arch. Biochem. Biophys. 294, 130-137, 1992) have reported the levels of plasma 3DG in normoglycemics to be 61 nM, using a GC/MS procedure. In contrast to this, Niwa et al. (Biochem. Biophys. Res. Commun. 196, 837-843, 1993) reported 3DG levels to be 1800 nM in normoglycemics, using a totally independent GC/MS method. To resolve this disagreement and fill the need for a robust assay for this dicarbonyl, suitable for absolute quantitation, a GC/MS procedure was devised for its measurement. Plasma samples were deproteinized either by ultrafiltration or by addition of ethanol as described by Niwa et al. (Biochem. Biophys. Res. Commun. 196, 837-843, 1993). 3DG in the ultrafiltrate or the supernatant was conjugated with 2,3-diamino-naphthalene to produce a stable adduct which was then converted to a silyl ether and analyzed by GC/MS. The analyte was monitored by selected ion monitoring at an m/z of 295 and 306 and quantitated using an internal standard of [U-13C]3DG. Using this approach, 3DG levels in plasma deproteinized by ultrafiltration were found to be significantly elevated from 58.5 +/- 14 (SD) nM in normoglycemics to 98.5 +/- 34 (SD) nM in type I diabetics. When deproteinization of the plasma was carried out using ethanol, the levels of 3DG from normoglycemic plasma were similar to those reported by Niwa et al. (1710 +/- 750 (SD) nM). These results suggest that 3DG levels measured by ultrafiltration may represent the free circulating 3DG and those obtained by ethanol extraction may represent aform of 3DG bound to a macromolecule (presumbaly protein).

Quantitation of resonances in biological 31P NMR spectra via principal component analysis: potential and limitations.

This paper examines the potential and limitations of peak area quantitation of biological NMR spectra using principal component analysis (PCA), including its requirement for prior knowledge. The principles of the method are presented without in-depth mathematical treatment. PCA is illustrated for simulated data, 31P NMR spectra obtained consecutively over 1-2.5 days from perfused Rat-2 cells metabolizing the choline analogue phosphoniumcholine (Chop) and in vivo proton-decoupled, NOE-enhanced, three-dimensional CSI localized 31P NMR spectra of the liver of healthy volunteers. The results show that PCA can be used to quantitate strongly overlapping peaks without prior knowledge of the peak shapes or positions and to reconstruct spectra with significantly reduced noise variance. Two major limitations of PCA are presented: (1) PCA cannot separate peaks whose intensities are well correlated; (2) PCA is sensitive to differences in chemical shift and line-width of peaks between spectra. The discussion focuses on what knowledge of the biological and spectroscopic features of the samples and the principles of PCA is necessary for peak area quantitation via PCA.

Identification of galactitol 2-phosphate and galactitol 3-phosphate in the lens of galactose-fed rats.

Production of unusual phosphorylated metabolites in the lens is one of several changes caused by hyperglycemia. Sorbitol 3-phosphate (Sor-3P) and fructose 3-phosphate (Fru-3P) are two such compounds identified in the diabetic lens, and galactitol 2-phosphate (Gal-2P) and galactitol 3-phosphate (Gal-3P) are identified here in the galactosemic lens. These new compounds are the first example of galactitol metabolism in mammalian tissue other than liver. Sor-3P and Fru-3P are also present in the galactosemic lens, apparently synthesized directly from their precursors, sorbitol and fructose, which are elevated in the lens due to increased flux of glucose through the aldose reductase (AR) pathway. The NADPH necessary to support this increased flux is derived from activation of the hexose monophosphate shunt (HMPS), which is clearly demonstrated by a large increase in the concentration of sedoheptulose 7-phosphate (Sed-7P), a HMPS-specific metabolite. Additionally, during 3 weeks of galactose feeding, there is a dramatic increase in lenticular concentrations of galactitol, sorbitol, galactose, and fructose and a sharp decrease in inositol. Glucose remains unchanged. A precipitous loss of both phosphorylated and nonphosphorylated metabolites occurs after 3 weeks, possibly due to lens rupture.

31P-nuclear magnetic resonance evidence of an activated hexose-monophosphate shunt in hyperglycemic rat lenses in vivo.

Using 31P-nuclear magnetic resonance spectroscopy, we have identified elevated concentrations of sedoheptulose-7-phosphate (S-7-P) in lenses from three animal models of hyperglycemia: streptozotocin-induced diabetic rats, galactose-fed rats, and xylose-fed rats. This observation provides a unique and independent confirmation of the activation of the hexose monophosphate shunt (HMPS) pathway in the hyperglycemic lens in vivo. While the elevation in concentration of S-7-P was very dramatic, the other HMPS metabolites in these tissues were below the threshold of detection, as expected for the HMPS pathway near equilibrium. In terms of nonenzymatic glycation, these results suggest that the only HMPS metabolite of importance in the hyperglycemic rat lens is S-7-P. Although in the diabetic lens its role appears to be relatively minor, in the galactosemic lens this compound may be an important contributor to the increased production of advanced glycosylation end products.

Metabolism of fructose-3-phosphate in the diabetic rat lens.

Fructose-3-phosphate and sorbitol-3-phosphate are produced in diabetic rat lenses by a 3-phosphokinase. While sorbitol-3-phosphate appears to be an inert polyol phosphate, fructose-3-phosphate is a potent cross-linking agent and a potential in vivo source of 3-deoxyglucosone. The objective of this study was to investigate the production and decomposition of fructose-3-phosphate in the diabetic rat lens. The results indicate that this metabolite achieves a steady-state concentration of almost 1 mumol/g wet weight within 2 weeks after the onset of diabetes. These steady-state levels appear to be a consequence of a balance between its production from fructose and its further decomposition to 3-deoxyglucosone. This conclusion is supported by results from disappearance of fructose-3-phosphate in insulin-treated diabetic rats and in vitro incubations of fructose-3-phosphate with amines where production of 3-deoxyglucosone was detected using a number of different methods including mass spectrometry. In agreement with these results, elevated concentrations of 3-deoxyglucosone along with its detoxification product, 3-deoxyfructose, were detected in the diabetic rat lenses. Other sugars and sugar phosphates which were detectable in the diabetic rat lenses were glucose, fructose, glucose-6-phosphate, fructose-6-phosphate, and sedoheptulose-7-phosphate. In conclusion, results from this study suggest that fructose-3-phosphate and 3-deoxyglucosone are likely to be important contributors to the process of nonenzymatic glycation in diabetic rat lenses.

Production of fructose and fructose-3-phosphate in maturing rat lenses.

PURPOSE: A large increase in glycation of crystallins between 1 and 8 months has been demonstrated in lenses obtained from aging rats. The objective of this study was to investigate if an age-associated increase in the levels of any of the phosphorylated and nonphosphorylated sugars in the aging rat lenses could be correlated with this increase. METHODS: Lenses were obtained from Sprague-Dawley rats ranging in age from 2 to 20 months. Trichloroacetic extracts of these tissues were analyzed by using 31P-NMR for sugar phosphates and high-pressure liquid chromatography equipped with an electrochemical detector for sugars and polyols. RESULTS: Although no elevation in the lenticular glucose levels was observed, an age-associated increase in the concentrations of polyol pathway-associated metabolites--sorbitol, fructose, sorbitol-3-phosphate, and fructose-3-phosphate--was detected. In contrast, no significant changes were observed in glycolytic or pentose shunt metabolites. CONCLUSION: Aging lenses accumulate increased concentrations of fructose and fructose-3-phosphate. Because fructose-3-phosphate is a potent glycating agent and a potential in vivo source of 3-deoxyglucosone, its accumulation in the lens, along with fructose, may be a contributing factor in the age-associated increase of nonenzymatic glycation in rat lenses.

Properties of fructose-1,6-bisphosphate aldolase from Escherichia coli: an NMR analysis.

A class II Zn(2+)-dependent fructose-1,6-bisphosphate (FBP)- aldolase was purified from an overproducer strain of Escherichia coli and characterized by standard biochemical techniques and 13C NMR spectroscopy. The principal finding of these studies was identification, by 13C NMR spectroscopy, of an enzyme-bound reaction intermediate, the enediol(ate) form of dihydroxyacetone phosphate (DHAP). Formation of this intermediate requires the presence of Zn2+ and is pH dependent, with increasing amounts of this tautomer appearing at alkaline pH's. This pH dependence closely parallels the pH activity profile of the enzyme, suggesting an involvement of the enediol-DHAP form in the reaction pathway. In addition to these results the following observations were made on this enzyme: (a) E. coli FBP aldolase binds and utilizes only the carbonyl forms of FBP and DHAP; (b) the function of Zn2+ in this metalloaldolase appears to be polarization of the C = O bond of DHAP; (c) activity of this enzyme is unaffected by glycolytic intermediates or nucleotide phosphates such as ATP. Although these studies provide some information about the catalytic mechanism of E. coli FBP aldolase, they do not provide an explanation for the apparent regulation of this enzyme reported in previous in vivo NMR studies. While the possibility that the enzyme is allosterically regulated cannot be excluded at this time, an interesting possibility suggested by this and other studies is that in E. coli glycolytic substrates may be channeled through a multienzyme complex.

Characterization of a phosphonium analog of choline as a probe in 31P NMR studies of phospholipid metabolism.

Tumors and transformed cells have been shown by 31P NMR to contain elevated concentrations of two phosphomonoesters, phosphorylcholine and phosphorylethanolamine, involved in phospholipid metabolism. In order to understand the biochemical basis for these phenomena new methods are needed to allow for analysis of the relevant metabolic pathways in intact cells. One such promising tool may be phosphonium-choline, a 31P NMR-visible analog of choline in which the trimethyl-ammonium group of choline has been replaced with a trimethyl-phosphonium moiety. As shown previously [Sim et al. Biochem. J. 154, 303 (1976)], this compound is non-toxic and readily metabolized by cultured cells into phospholipids. In this paper we describe in greater detail some of the chemical and NMR spectroscopic properties of this material. Most significantly we show here that the chemical shift of phosphonium-choline is sensitive to the phosphorylation state of the analog and that the phosphonium nucleus is NMR-visible even after its incorporation into phospholipid. The unique properties of this analog should make it possible to use high-field 31P NMR to follow the flux of phosphonium-choline through the Kennedy pathway in intact perfused cells cultures.

Effect of pH on the bioenergetics of perfused porcine lenses.

Extracellular and intracellular pH has been shown to be an important physiological variable in many biological systems. However, studies on the effect of pH on the metabolic status of the mammalian lens have been few. It has been shown previously that a change in perfusate pH has a significant effect on the lens weight, ion transport, opacification and membrane potential. In this study we show that the bioenergetic status of the perfused pig lens, as assessed by 31P-NMR spectroscopy, is critically dependent on the pH of the bathing medium. At pH's of 7.6 and above, these perfused lenses maintained their nucleotide triphosphates concentrations steady for up to 4 days. In contrast, lenses perfused with identical media at pH 7.0, rapidly lost their nucleotide triphosphates. Our findings suggest that the pH of the extracellular pH may be an important parameter in maintaining the functional competence of the lens.

Detection of fructose-3-phosphokinase activity in intact mammalian lenses by 31P NMR spectroscopy.

Recently we have identified two novel phosphorylated metabolites in the lenses of diabetic rats as sorbitol 3-phosphate (Sor-3-P) and fructose 3-phosphate (Fru-3-P). The latter compound is of particular interest since it is a potent glycating agent, which could account, at least in part, for the increased protein cross-linking and cataract formation in the lens of the diabetic rat. In order to gain insight into the mechanism of formation of these compounds, 31P NMR spectra of rat, pig, and rabbit lenses, perfused with media supplemented with glucose, fructose, or sorbitol, were acquired. Perfusion with fructose-supplemented media resulted in the production of Fru-3-P in all three species. This compound was not produced upon perfusion with fructose-deficient media. The identification of the newly synthesized material as Fru-3-P was confirmed by spiking perchloric acid extracts of the perfused lenses with synthetic Fru-3-P. Our results provide strong evidence for the existence of a fructose-3-phosphokinase in mammalian lenses. If this enzyme is present in human lenses as well, it will reinforce the hypothesis that this enzyme and its product, Fru-3-P, may play a role in diabetic cataractogenesis and that lenticular fructose-3-phosphokinase may provide another therapeutic target in the prevention and alleviation of diabetic cataracts.