Effect of proton pump inhibitors on Magnesium levels in Type II diabetic patients: a single centre study from Saudi Arabia.

OBJECTIVE: In recent years, increasing evidence suggests an association between low Magnesium levels and type 2 diabetes mellitus. It has also been reported that the use of proton pump inhibitors may induce hypomagnesemia. Although some case reports have described patients with Proton Pump Inhibitor-induced hypomagnesemia, the impact of Proton Pump Inhibitor use on hypomagnesemia has not been fully clarified in comparative studies. The objective of the study was to determine the Magnesium levels in patients with diabetes who are taking proton pump inhibitors and also to correlate the Magnesium levels in diabetic patients who take proton pump inhibitors with those not taking proton pump inhibitors. PATIENTS AND METHODS: A cross-sectional study was carried out in the study population comprising adult patients attending internal medicine clinics in King Khalid Hospital, Majmaah, KSA. A total of 200 patients who gave informed consent were recruited into the study over one year. RESULTS: Overall prevalence of hypomagnesemia was observed among 128 patients out of 200 (64%) diabetic patients. Relatively more patients with hypomagnesemia were found in group 2 (without PPI use) (38.5%) compared to group 1 (with PPI use) (25.5%). A statistically significant difference was not observed in group 1 using proton pump inhibitors and group 2 not using proton pump inhibitors (p-value = 0.473). CONCLUSIONS: Hypomagnesemia is seen in diabetic patients and patients who take proton pump inhibitors. There was no statistically significant difference in Magnesium levels in diabetic patients, irrespective of proton pump inhibitor use.

Metabolism of dietary ODAP in humans may be responsible for the low incidence of neurolathyrism.

OBJECTIVES: The reasons for the very low incidence of the disease neurolathyrism in humans even after excessive consumption of the pulse, Lathyrus sativus, under severe drought and famine conditions, and its continued consumption by large populations during normal periods without any deleterious effects have been examined in the context of a possible metabolism or detoxification of beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP), the major neurotoxic amino acid of L. sativus. DESIGN AND METHODS: ODAP in urine samples from 54 subjects habitually consuming the pulse and in three volunteers on an L. sativus diet was determined by the OPT method following clean up of the samples on an alumina column. Urinary oxalate was also determined in these individuals. RESULTS: Twenty-five subjects showed no excretion of ODAP and it was only less than 0.7% of the dietary intake in the remaining 29 subjects. Urinary excretion of ODAP in three volunteers was also less than 1% with a peak excretion in the 4-h sample. The 4-h blood sample from one volunteer had a maximum ODAP concentration of 177 microM. The urinary oxalate content in the volunteers was nearly 3-fold higher compared to controls. CONCLUSIONS: The low excretion of dietary ingested ODAP in humans is in sharp contrast to that seen in animals and indicates a metabolism or detoxification of ODAP which may be unique to humans and may explain the low incidence of neurolathyrism.

Specific substrate for CLN2 protease/tripeptidyl-peptidase I assay.

The classic late infantile neuronal ceroid lipofuscinosis (LINCL, CLN2) is a fatal neurodegenerative disorder that results from mutations in a gene encoding a lysosomal proteinase, known as CLN2 protease (CLN2p) or tripeptidyl peptidase I (TPP-I). Three different substrates, fluorescein isothiocyanate-labelled haemoglobin, A-F-F-7-amino-4-methylcoumarin (AAF-AMC) and G-F-F-L-7-amino-4-trifluoromethylcoumarin (GFFL-AFC) have been used for the CLN2p/TPP-I assay with varying degrees of residual activities in patients with LINCL. Further, conclusive identification of carriers are not possible with the first two substrates. An assay for the CLN2p/TPP-I based on the cleavage of amino terminal tripeptide from G-F-F-L-AFC was applied to prenatal and postnatal diagnosis of LINCL patients and heterozygote carriers. In leukocytes, the CLN2p/TPP-I activities in controls and heterozygote carriers were 1995 +/- 154 (n = 15) and 918 +/- 253 (n = 15) nmol/h/mg protein respectively. No CLN2p/TPP-I activity was detectable in all but two patients. These two patients had less than 2% residual activity, and had delayed clinical symptoms for LINCL. This shows that the G-F-F-L-AFC is a highly specific substrate for the CLN2p/TPP-I assay. The fact that with this substrate the enzyme cleaves a peptide bond between the two amino acids may be the reason for the high level of specificity.

Biochemistry of neuronal ceroid lipofuscinoses.

This chapter summarizes the recent advances that have been made with respect to biochemical characterization of the neurodegenerative diseases collectively known as neuronal ceroid lipofuscinoses (NCL) or Batten disease. Genomic and proteomic approaches have presently identified eight different forms of NCL (namely, CLN1 through CLN8) based on mutations in specific genes. CLN1 and CLN2 are caused by mutations in genes that encodes lysosomal enzymes,palmitoyl protein thioesterase and pepstatin-insensitive proteinase, respectively. The protein involved in the etiology of CLN3 is a highly hydrophobic, presumably transmembrane protein. NCL are considered as lysosomal storage diseases because of the accumulation of autofluorescent inclusion bodies. The composition of inclusion bodies varies in different forms of the NCL. The major storage component in CLN2 is the subunit c of mitochondrial ATP synthase complex and its accumulation is the direct result of lack of CLN2p in this disease. Mannose-6-phosphorylated glycoproteins accumulate in CLN3 and most likely their accumulation is the result of an intrinsic activity of the CLN3 protein. Significant levels of oligosaccharyl diphosphodolichol also accumulate in CLN3 and CLN2, whereas lysosomal sphingolipid activator proteins (saposins A and D) constitute major component of the storage material in CLN 1. The issue of selective loss of neuronal and retinal cells in NCL still remains to be addressed. Identification of natural substrates for the various enzymes involved in NCL may help in the characterization of the cytotoxic factor(s) and also in designing rationale therapeutic interventions for these group of devastating diseases.

Proteomic approach for the elucidation of biological defects in autism.

Proteomic-based approaches, which examine expressed proteins in tissues or cells, have great potential in the elucidation of biological defects in heterogeneous neurodevelopmental disorders such as autism. In this approach, tissue or cellular proteins from control and affected subjects are separated on two-dimensional (2-D) polyacrylamide gel electrophoresis, and those proteins that show marked changes in the concentration between control and affected subjects are identified by mass spectroscopy. This method has been successfully applied in the elucidation of the molecular biological defect in classic late-infantile neuronal ceroid lipofuscinosis (Sleat et al., 1997). Unlike the classical methods of genome-wide screening for chromosomal localization followed by positional cloning, the proteomic approach requires limited number of tissue samples and the study can be completed in a relatively short time. Currently, these methods are available for relatively abundant proteins and generally are not applicable for hydrophobic proteins because 2-D gel electrophoresis is not very effective in the analysis of hydrophobic proteins. The genetic defect results in either total loss of proteins or changes in molecular weight and/or isoelectric point will be detectable by the proteomic method. Because autism is a neurogenetic disorder, brain is the tissue of choice for proteomic study. For an oligogenic disorder such as autism, at least some of the aberrant (genes) proteins may be identified by this technology.

A lysosomal pepstatin-insensitive proteinase as a novel biomarker for breast carcinoma.

Lysosomal proteinases play an important role in the turnover of intracellular proteins, and acidic proteinases such as cathepsin D are known to be increased in breast carcinoma. In the present study the activity of a newly discovered acidic lysosomal pepstatin-insensitive proteinase (CLN2p) was measured in breast tissues by the most sensitive and highly specific assay that we had developed for the diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) (2). Samples from eight normal subjects undergoing reductive mammoplasty and 200 patients with primary breast carcinoma were analyzed. The results suggest a two- to seventeen-fold higher CLN2p activity in tumors, which was significantly and positively correlated with already known breast cancer biomarkers such as levels of cathepsin D, estrogen receptor and progesterone receptor. These results suggest a diagnostic and prognostic potential for this novel acid proteinase in breast cancer.

Purification and characterization of bovine brain lysosomal pepstatin-insensitive proteinase, the gene product deficient in the human late-infantile neuronal ceroid lipofuscinosis.

A lysosomal pepstatin-insensitive proteinase (CLN2p) deficiency is the underlying defect in the classical late-infantile neuronal ceroid lipofuscinosis (LINCL, CLN2). The natural substrates for CLN2p and the causative factors for the neurodegeneration in this disorder are still not understood. We have now purified the CLN2p from bovine brain to apparent homogeneity. The proteinase has a molecular mass of 46 kDa and an aminoterminal sequence, L-H-L-G-V-T-P-S-V-I-R-K, that is identical to the human enzyme. Peptide: N-glycosidase F and endoglycosidase H treatment of the CLN2p reduced its molecular mass to 39.5 and 40.5 kDa, respectively, suggesting the presence of as many as five N-glycosylated residues. The CLN2p activity was not affected by common protease inhibitors, and thiol reagents, metal chelators, and divalent metal ions had no significant effect on the proteolytic activity of the CLN2p. Among the naturally occurring neuropeptides, angiotensin II, substance P, and beta-amyloid were substrates for the CLN2p, whereas angiotensin I, Leu-enkephalin, and gamma-endorphin were not. Peptide cleavage sites indicated that the CLN2p is a tripeptidyl peptidase that cleaves peptides having free amino-termini. Synthetic amino- and carboxyl-terminal peptides from the subunit c sequence, which is the major storage material in LINCL, are hydrolyzed by the CLN2p, suggesting that the subunit c may be one of the natural substrates for this proteinase and its accumulation in LINCL is the direct result of the proteinase deficiency.

Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected].

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.

Neurotoxic potential of three structural analogs of beta-N-oxalyl-alpha,beta-diaminopropanoic acid (beta-ODAP).

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of beta-ODAP were synthesized. Carboxymethyl-alpha,beta-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 microM), but not by CNQX (10 microM). N-acetyl-alpha,beta-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three beta-ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2-3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [3H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for beta-ODAP. These data indicate that analogs of beta-ODAP may interact with glutamate receptors without producing neurotoxicity.

Increased brain lysosomal pepstatin-insensitive proteinase activity in patients with neurodegenerative diseases.

A recent study has shown mutations in CLN2 gene, that encodes a novel lysosomal pepstatin-insensitive proteinase (LPIP), in the pathophysiology of late-infantile neuronal ceroid lipofuscinosis (LINCL). We have measured the LPIP activities in brains from various forms of human neuronal ceroid lipofuscinoses (NCL), canine ceroid lipofuscinosis and other neurodegenerative disorders with a highly sensitive assay using a tetrapeptide Gly-Phe-Phe-Leu-amino-trifluoromethyl coumarin (AFC) as substrate. Brain LPIP has a pH optimum of 3.5 and an apparent km of 100 microM for the crude enzyme. The enzyme activity is totally absent in LINCL patients. Pronounced increase in the LPIP activity was seen in patients suffering from infantile (INCL), juvenile (JNCL) and adult (ANCL) forms of neuronal ceroid lipofuscinoses. LPIP activity was also found to be increased about two-fold in Alzheimer's disease when compared with normal or age-matched controls, while in globoidal-cell leukodystrophy (Krabbe's disease) it was similar to the normal controls. Although mannose-6-phosphorylated LPIP is increased 13-fold in brains of patients with JNCL, this form of LPIP did not have any enzyme activity. The mechanism by which LPIP activities are increased in a wide range of neurodegenerative diseases is unknown, although neuronal loss, followed by gliosis are common characteristics of these diseases.

A novel assay for lysosomal pepstatin-insensitive proteinase and its application for the diagnosis of late-infantile neuronal ceroid lipofuscinosis.

A highly sensitive assay for mammalian lysosomal pepstatin-insensitive proteinase (LPIP) is described using a synthetic peptide substrate coupled to aminotrifluoromethyl coumarin (AFC). LPIP is an endocarboxyl proteinase which has specific sequence requirements of Phe-Phe around the carboxyl terminal. This HPLC based assay can detect patients suffering from late-infantile neuronal ceroid lipofuscinosis (LINCL) and also heterozygote carriers in cultured lymphoid cells and skin fibroblasts. None of the patients analyzed had detectable enzyme activity confirming the defective gene product, while carriers had about 50% activity when compared with the normal controls. Neurological controls comprised of patients with other neurodegenerative disorders have LPIP activities similar to normal controls. LPIP activity is also detectable in amniocytes and chorionic villi. Thus the assay reported can also be used for prenatal diagnosis of LINCL.

Receptor interactions of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, the Lathyrus sativus putative excitotoxin, with synaptic membranes.

Direct evidence for the excitotoxicity of 3-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 3H]ODAP ([3H]ODAP) to synaptic membranes. [3H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15-20% of the total binding) and was also not saturable. The low specific binding of [3H]ODAP remained unaltered under a variety of assay conditions. A low Bmax of 3.2 +/- 0.4 pmol/mg and Kd 0.2 +/- 0.08 microM could be discerned for the high affinity interactions under conditions wherein more than 80-90% of the binding was nonspecific. While ODAP could inhibit the binding of [3H]glutamate to chick synaptic membranes with a Ki of 10 +/- 0.9 microM, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [3H]glutamate. The very low specific binding of [3H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.

The distribution of total, free, short-chain acyl and long-chain acyl carnitine in ocular tissues of the camel (Camelus dromedarius).

Carnitine (CA) plays an important role in the metabolism of fatty acids. Its presence is considerable in tissues that utilize fatty acids as an important source of energy, such as the heart and the skeletal muscle. The presence of free, short-chain acyl and long-chain acyl carnitine was shown for the first time in various tissues of the camel eye. The ratio of acyl carnitine (AC) to free carnitine (FC) was high in aqueous humor, indicating a predominance of AC, while it was low in vitreous humor, indicating a higher concentration of FC. The highest concentration of total carnitine (TC) was observed in the retina, followed by the cornea, while the lowest was found in lens nucleus. Among various forms, FC concentration was the highest in the cornea while it was the lowest in the lens cortex. In the cornea and retina, the proportion of short-chain acylcarnitines (SC) was two-fold when compared to long-chain acylcarnitines (LC), while in the lens it was the same. The ratio of AC to FC in the retina was high, indicating a predominance of AC, while in lens it was low, indicating a higher concentration of FC. In contrast, L-CA in the cornea was present equally between the free and the esterified forms. The quantitative differences in the distribution of L-CA in various tissues of the camel eye reflect that it may be involved in providing a source of readily available energy.

Purification of carnitine acetyltransferase from skeletal muscle of the camel (Camelus dromedarius).

The enzyme carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short-chain acyl groups between coenzyme A and L-carnitine, and hence, plays an important role in the beta-oxidation of fatty acids. Purification and characterization of CAT from desert animal species may help in explaining the involvement of secondary pathways for energy production in these species. In this paper, we report the purification and partial characterization of CAT from the Arabian camel. CAT was purified from the skeletal muscle of the Arabian camel by ammonium sulfate and acetone fractionation, followed by chromatography on DEAE-Sepharose, agarose-Co A and Superose 12 gel filtration columns. CAT was purified by 2937-fold to a specific activity of 94 Units mg-1. The purified CAT was a monomer of 59 kDa as judged by native and SDS-PAGE, and showed a pI of 5.2. The enzyme displayed maximum activity with propionyl-Co A. Apparent Km for acetyl-, propionyl- and butyryl-Co A were 27.7, 17.3 and 29 microM respectively, while palmitoyl-Co A was not a substrate.

Investigation of the influence of theophylline feeding on total, free, short-chain acyl and long-chain acyl carnitine levels in skeletal muscle and liver of rats.

L-carnitine, the obligate fatty acyl carrier across the inner mitochondrial membrane, is sensitive to biochemical changes that alter lipid metabolism. The present study describes the effects of oral theophylline on L-carnitine concentrations in the skeletal muscle and the liver of rats. A significant rise in total, free and long-chain acyl carnitine concentrations was observed in the skeletal muscle but not in the liver. The increases observed after the first week of theophylline feeding returned to the control concentrations after fifth week despite the continuation of treatment. The observed changes in the skeletal muscle may be due to a) increased acyl carnitines that are formed as a consequence of enhanced fatty acid mobilization and b) increased L-carnitine uptake by the tissue.

Kinetic properties of purified carnitine acetyltransferase from the skeletal muscle of Arabian camel (Camelus dromedarius).

The kinetic properties of carnitine acetyltransferase from the skeletal muscle of the Arabian camel (Camelus dromedarius) were studied. The enzyme showed an optimum pH between 7.2 and 8.2. Reciprocal plots of data obtained by varying one substrate concentration while keeping the other constant revealed lines that converged on the abscissa, indicating that the enzyme possible follows a random mechanism of catalysis. The Kms for L-carnitine and acetyl-coenzyme A were 244 and 44 microM respectively, while those for acetyl-DL-carnitine and coenzyme A (Co A) were 307 and 39 microM respectively. The Km for one substrate was found to be independent of the concentration of the second substrate used. Corresponding Vmax values for L-CA, acetyl-Co A, acetyl-DL-carnitine and Co A are 98, 98, 102 and 100 mumol min-1 mg-1 protein respectively. The low Km obtained for acetyl-DL-carnitine suggests an adaptive mechanism in this desert species for enduring prolonged dry spells without food and water.

Determination of L-carnitine, acylcarnitine and total carnitine levels in plasma and tissues of camel (Camelus dromedarius).

The total, free and acyl carnitine levels were measured in plasma and tissues of the Arabian camel (Camelus dromedarius). Significant variation in carnitine concentrations were observed in plasma and tissues of the camel when compared with other animal species. A higher proportion of acyl carnitine was found in plasma and skeletal muscle of the camel than other animal species. Among the camel tissues, skeletal muscle possessed the highest amount of carnitine while the lowest amount was found in kidney. The higher carnitine content and a higher proportion of acyl carnitine in plasma and tissues of the Arabian camel suggest an adaptive mechanism that could be common to desert animal species.