[Progress on the Pathophysiology of Idiopathic Basal Ganglia Calcification].

Idiopathic basal ganglia calcification (IBGC), which is also called Fahr's disease or recently referred to as primary familial brain calcification (PFBC), is an idiopathic and intractable disease characterized by abnormal deposits of minerals including calcium in the basal ganglia and other brain regions such as the thalamus and cerebellum. Mutations in SLC20A2, PDGFRB, PDGFB, XPR1, MYORG have been reported in the past several years. The pathophysiological basis presumed by the genetic studies is the impairment of the transport of inorganic phosphate (Pi) into and out of cells in the brain. We reported high levels of Pi in the cerebrospinal fluid (CSF) of IBGC patients, especially in IBGC patients with SLC20A2 mutations. The flow of Pi between the CSF and interstitial fluid (ISF) in the brain and the drainage flow through the perivascular space in the perivascular drainage pathway can explain the distribution and pathology of mineralization in IBGC. Thus, it is very important to further elucidate the pathophysiology of IBGC and consequently develop pharmacological agents based on the pathophysiology of IBGC in the near future in order to benefit patients with IBGC and their families.

SLC20A2 Deficiency in Mice Leads to Elevated Phosphate Levels in Cerbrospinal Fluid and Glymphatic Pathway-Associated Arteriolar Calcification, and Recapitulates Human Idiopathic Basal Ganglia Calcification.

Idiopathic basal ganglia calcification is a brain calcification disorder that has been genetically linked to autosomal dominant mutations in the sodium-dependent phosphate co-transporter, SLC20A2. The mechanisms whereby deficiency of Slc20a2 leads to basal ganglion calcification are unknown. In the mouse brain, we found that Slc20a2 was expressed in tissues that produce and/or regulate cerebrospinal fluid, including choroid plexus, ependyma and arteriolar smooth muscle cells. Haploinsufficient Slc20a2 +/- mice developed age-dependent basal ganglia calcification that formed in glymphatic pathway-associated arterioles. Slc20a2 deficiency uncovered phosphate homeostasis dysregulation characterized by abnormally high cerebrospinal fluid phosphate levels and hydrocephalus, in addition to basal ganglia calcification. Slc20a2 siRNA knockdown in smooth muscle cells revealed increased susceptibility to high phosphate-induced calcification. These data suggested that loss of Slc20a2 led to dysregulated phosphate homeostasis and enhanced susceptibility of arteriolar smooth muscle cells to elevated phosphate-induced calcification. Together, dysregulated cerebrospinal fluid phosphate and enhanced smooth muscle cell susceptibility may predispose to glymphatic pathway-associated arteriolar calcification.

Molecular architecture of Aß fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aß plaque formation.

OBJECTIVES: The detailed structure of brain-derived Aß amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aß(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aß amyloid plaque formation. METHODS: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aß fibrils. RESULTS AND CONCLUSIONS: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aß peptide fold is common to Aß fibrils.

Slc20a2 is critical for maintaining a physiologic inorganic phosphate level in cerebrospinal fluid.

Mutations in the SLC20A2-gene encoding the inorganic phosphate (Pi) transporter PiT2 can explain approximately 40% of the familial cases of the rare neurodegenerative disorder primary familial brain calcification (Fahr's disease). The disease characteristic, cerebrovascular-associated calcifications, is also present in Slc20a2-knockout (KO) mice. Little is known about the specific role(s) of PiT2 in the brain. Recent in vitro studies, however, suggest a role in regulation of the [Pi] in cerebrospinal fluid (CSF). We here show that Slc20a2-KO mice indeed have a high CSF [Pi] in agreement with a role of PiT2 in Pi export from the CSF. The implications in relation to disease mechanism are discussed.

Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent.

We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5'-triphosphate (UTP), was produced by MTX-treated Hela cells.

Induction of a phosphate appetite in adult male and female rats.

We have reported that dietary inorganic phosphate (Pi) deprivation induces a Pi-seeking behavior in juvenile male rats. The purpose of the present study was to determine whether the Pi appetite is present in adult animals, and if so, whether it is altered during times of increased demand for Pi, such as pregnancy and lactation. Both male and female animals fed a low-phosphate diet (LPD) ingested significantly greater amounts of PiH(2)O daily than their normal phosphate diet (NPD) controls, and per 100 g of body weight (BW), the female animals fed LPD tended to ingest greater amounts of PiH(2)O than male rats fed LPD. Pregnant and lactating rats fed LPD ingested significantly more PiH(2)O than those fed NPD, however, neither group displayed a Pi appetite different than virgin females. However, lactation further reduced Pi levels in plasma and cerebral spinal fluid compared with control values. Despite the additional Pi from the PiH(2)O in the mothers fed LPD, pup birth weight was significantly lower than in NPD litters, and this was exacerbated 9 days after birth. This attenuated BW gain was associated with lower plasma Pi levels in the pups. In conclusion, a mild but consistent Pi-seeking behavior is induced in adult male and female rats after only 2 days of dietary Pi restriction. On a relative basis, the amount of PiH(2)O ingested is greater in female than in male animals, but does not increase further during pregnancy and lactation.

Central control of renal sodium-phosphate (NaPi-2) transporters.

Regulation of phosphate (Pi) reabsorption occurs through the up- and downregulation of the renal type-II sodium Pi cotransporters (NaPi-2). Recently, renal NaPi2-type expression has been identified in areas of the brain. The present study determined whether brain NaPi-2 is regulated by dietary Pi and whether the behavioral and renal adaptations to low-dietary Pi are controlled centrally. NaPi-2-like expression in the third ventricle (3V) and amygdala of juvenile Wistar rats was regulated by dietary Pi, as in the kidneys. When cerebrospinal fluid (CSF) Pi concentration was elevated by 3V injections of Pi in rats fed low-Pi diet (LPD), the behavioral and renal adaptations to LPD were abolished. Most importantly, NaPi-2 expression was markedly reduced not only in the brain, but also renal proximal tubules, despite the low plasma Pi milieu. This was confirmed by the significant reduction in the transport maximum for Pi (from 8.1+/-0.2 in LPD + veh 3V to 1.7+/-0.1 micromol Pi/ml glomerular filtration rate in LPD + 3V Pi, P < 0.001). These findings indicate that NaPi-2-like transporters in the brain are regulated by both dietary Pi and CSF Pi concentrations, and most significantly, that the central Pi milieu can regulate renal NaPi-2 expression. We hypothesize that central 3V NaPi-2 transporters may act as Pi sensors and help regulate both brain and whole body Pi homeostasis.

Evidence for induction of a phosphate appetite in juvenile rats.

This study examined whether dietary phosphate (Pi) restriction stimulates an appetite for Pi in the juvenile rat, which normally has a high metabolic Pi demand for growth. Juvenile Wistar rats were placed in individual cages with unrestricted access to tap water and a low (LPD, 0.02% Pi) or normal Pi diet (NPD, 0.6% Pi) for 7 days. On day 8, both groups of rats were given unlimited access to a solution of 0.3 M potassium phosphate water (PiH2O) for 8 additional days. Rats fed LPD consumed 70-100% more PiH2O then those rats fed NPD (P < 0.001). The increase in PiH2O intake resulted in a marked rise in the growth rate of rats fed LPD during days 8-15. A similar Pi intake was inducible after only 2 days of LPD and was associated with significant reductions in both plasma and cerebrospinal fluid (CSF) Pi levels; these levels remained low throughout Pi restriction, despite a significant PiH2O intake. Furthermore, the renal adaptation to enhance Pi reabsorption (TmPi) during Pi deprivation remained elevated despite enhanced PiH2O intake. Replenishment with a high-Pi diet rapidly quenched the PiH2O appetite and was associated with restoration of both plasma and CSF Pi levels. These findings suggest that an appetite for Pi can be induced in juvenile rats, perhaps through lowered plasma and CSF Pi levels. This behavioral response may serve as an additional mechanism to maintain an adequate supply of Pi necessary for growth and development of the animal.

Electrolyte levels in the CSF of children with nontumoral hydrocephalus. Relation to clinical parameters.

The levels of calcium and magnesium (by atomic absorption spectrophotometry), sodium and potassium (by flame photometry), inorganic phosphate and proteins (by spectrophotometry) were measured in the CSF of 27 children with hydrocephalus and compared with the CSF data on 10 controls, so as to study the effect of the blood-brain and blood-CSF barrier alterations observed in hydrocephalus, on the CSF electrolytes. Ca (P less than 0.001), Mg (P less than 0.01) and phosphate (P less than 0.05) were found to be increased, K (P less than 0.01) decreased, and Na levels not significantly altered. Linear regression analysis of hydrocephalic patients showed a linear correlation (r = +0.62) between phosphate and proteins (P less than 0.01) and (r = +0.66) between phosphate and Ca (P less than 0.001). Comparison of control CSF electrolyte levels with those found in the CSF of hydrocephalic patients were grouped according to clinical variables: sex (F, M), age (less than or equal to 1.5 months, greater than 1.5 months), etiological variants (congenital, acquired), treated infection (no, yes), surgical treatment (no, yes), time of onset (less than or equal to 1 month, greater than 1 month), ventricular dilation (mild, severe) and intracranial hypertension (mild or severe). These comparisons showed specific increases of Mg for males (P less than 0.05), acquired hydrocephalus (P less than 0.05) and severe intracranial hypertension (ICH) (P less than 0.001), moderate decrease in the Na levels in acquired hydrocephalus (P less than 0.05) and mild ICH (P less than 0.05), specific increases of phosphate in females (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[Reference values of sodium, potassium, chloride, calcium, inorganic phosphate and magnesium levels in the cerebrospinal fluid of children]. / Referenzwerte der Natrium-, Kalium-, Chlorid-, Kalzium-, anorganischen Phosphat- und Magnesium-Konzentration im Liquor cerebrospinalis von Kindern.

Concentration of sodium, potassium, chloride, calcium, inorganic phosphate and magnesium was determined in cerebrospinal fluid of 155 children (ages 1 month - 16 years). On the basis of clinical criterions the children were considered to be "healthy". The total number of examined children was 2 500 (1978-1983). The statistical age- and sexspecific investigation of the results showed no significant differences. Compilation of the values resulted in a physiological concentration of sodium 132.3 +/- 17.6 mval/l, potassium 2,59 +/- 0.37 mval/l, chloride 113.1 +/- 15.5 mval/l, calcium 3.47 +/- 1.45 mval/l, inorganic phosphate 1.11 +/- 0.21 mg/dl and magnesium 2.60 +/- 0.46 mg/dl.

Effects of 'DIDS', an anion transport blocker, on CSF [HCO3-] in respiratory acidosis.

During acute respiratory acidosis increments in cisternal cerebrospinal fluid (CSF) [HCO3-] approximate decrements in CSF [Cl-] with CSF [Na+] remaining unchanged; the mechanisms mediating this reciprocal anionic relationship are unclear. In the present study we investigated the effects of DIDS (4,4'-diisothiocyano-disulfonic stilbene), a known inorganic anion exchange blocker, on CSF ionic regulation in acute respiratory acidosis. In two groups of anesthetized paralyzed dogs we injected either mock CSF (group I, n = 8) or mock CSF containing DIDS (group II, n = 9) into the lateral cerebral ventricles. After 45 min, acute respiratory acidosis was induced for 6 h. During acute respiratory acidosis, CSF PCO2 rose in average by 38 mm Hg in both groups; increments in CSF [HCO3-], however, were significantly lower by about 2 mEq/L in DIDS-treated animals than in controls throughout the experimental period. Such differences were not due to changes in CSF lactate concentration which were similar in both groups. Furthermore, CSF [Na+] remained unchanged in both groups. Since disulfonic stilbene derivatives combine selectively with the carrier involved in anion transport and inhibit inorganic anion exchange, the data in the present study suggest that in the central nervous system a DIDS-inhibitable carrier is involved in the rise of CSF [HCO3-] observed during acute respiratory acidosis.

Effects of acetazolamide on ionic composition of cisternal fluid during acute respiratory acidosis.

We studied the effects of intravenous acetazolamide (50-200 mg/kg) on cerebrospinal fluid (CSF) electrolytes and pH regulation in 10 anesthetized and nephrectomized dogs (group II): acetazolamide was injected at -1 h, and respiratory acidosis was induced at zero time for 6 h. A control group of 10 animals (group I) was treated similarly except that an equal volume of 0.45% saline was injected intravenously instead of acetazolamide. The mean CSF PCO2 values in group I were 49.7 +/- 3.4 (SD), 50.2 +/- 3.6, 92.3 +/- 7.0, 100.3 +/- 8.1, and 97.8 +/- 7.3 Torr, respectively, at -1, 0, 3, 4.5, and 6 h; respective values in group II were 49.8 +/- 2.0, 55.2 +/- 5.2, 95.8 +/- 6.4, 103.1 +/- 16.7, and 104.9 +/- 14.1 Torr. During acute respiratory acidosis CSF [HCO3-] rose progressively with time in group I, and the mean values were 28.1 +/- 1.4 (SD), 29.2 +/- 1.7 and 30.1 +/- 1.9 mmol/l, respectively, 3, 4.5, and 6 h after induction of acidosis; respective values in group II were 28.2 +/- 1.1, 28.3 +/- 0.9, and 28.5 +/- 1.4 mmol/l. Acetazolamide at various doses administered inhibited any further rise in CSF [HCO3-] beyond the 3rd h of acidosis. The lower rise in CSF [HCO3-] in group II could not be ascribed to differences in CSF lactate concentration which changed similarly in both groups. Increments in CSF K+ and phosphate concentrations were significantly higher in the acetazolamide group than in the control group, the former presumably reflecting efflux of K+ from intracellular to extracellular fluid compartment. We conclude that in nephrectomized dogs during acute respiratory acidosis intravenously administered acetazolamide diminishes the rise in CSF [HCO3-], impairs CSF H+ regulation, and increases CSF K+ and phosphate concentrations.

Effects of SITS, an anion transport blocker, on CSF ionic composition in metabolic alkalosis.

Disulfonic stilbenes combine with the carrier protein involved in anion transport and inhibit the exchange of Cl- for HCO3- in a variety of biomembranes. Our aim was to determine whether such a mechanism is operative in the regulation of cerebrospinal fluid (CSF) [HCO3-] in metabolic alkalosis. In anesthetized, curarized, and artificially ventilated dogs either mock CSF (group I, 9 dogs) or mock CSF containing SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (group II, 7 dogs) was periodically injected into both lateral cerebral ventricles. During 6 h of isocapnic metabolic alkalosis, produced by intravenous infusion of Na2CO3 solution, plasma [HCO3-] was increased by approximately 14 meq/l in both groups. In SITS-treated animals the mean cisternal CSF [HCO3-] increased by 7.7 meq/l after 6 h, and this was significantly higher than the respective increment, 3.5 meq/l, noted in the control group. Increments in CSF [HCO3-] in both groups were reciprocated by decrements in CSF [Cl-] with CSF [Na+] remaining unchanged. Cisternal CSF PCO2 and lactate concentrations showed similar increments in both groups. It is hypothesized that in metabolic alkalosis a carrier transports HCO3- out of cerebral fluid in exchange for Cl- and that SITS inhibits this mechanism. The efflux of HCO3- out of CSF in metabolic alkalosis would minimize the rise in CSF [HCO3-] brought about by HCO3-] influx from blood into CSF and therefore contributes to the CSF [H+] homeostasis.

Immunoreactive calcitonin in cerebrospinal fluid of man.

We have detected immunoreactive calcitonin (iCT) in the cerebrospinal fluid (CSF) of normal individuals. Using an antibody with midportion recognition, the mean +/- S.D. of the cerebrospinal iCT in 27 normal subjects was 28 +/- 14 pg/ml. The mean serum iCT was 89 +/- 68 pg/ml, the CSF/serum distribution ratio being 0.31. There were no significant correlations between CSF iCT or serum iCT and the calcium, magnesium, phosphate, sodium, potassium or chloride in the CSF or serum. Although there was a trend for serum iCT values to be related to CSF iCT values, it did not attain statistical significance. The demonstration that the CSF contains iCT may have important physiologic implications, and its measurement offers a useful parameter to study its effects on calcium metabolism and/or other aspects of brain function.

Magnesium and inorganic phosphate content in CSF related to blood-brain barrier function in neurological disease.

In normal controls and in a large number of neurological patients divided into certain disease groups both Mg and PO4 were determined in cerebrospinal fluid (CSF) and serum. For both Mg and PO4 there was a marked concentration gradient between CSF and serum in normals where Mg was higher and PO4 content lower in CSF. Comparison of CSF values with serum values of patients showed pathological changes only in CSF, serum values always being within the control range. A number of disease processes associated with a disturbance of blood-brain barrier (BBB) function such as inflammatory CNS disease or CNS tumors showed significant alterations of PO4 concentrations in CSF which are interpreted as an approximation of serum values. A similar decrease of Mg did not reach statistical significance. Both Mg and PO4 in CSF showed a correlation with CSF protein concentrations, but no relationship with cells in CSF. Patients with cerebrosvascular disease were not significantly different from controls as regards their Mg and PO4 in CSF, but a small subgroup consisting of patients with an intracranial hemorrhage showed elevation of both Mg and PO4 which could signify cell necrosis rather than BBB dysfunction. Patients with disc protrusion or peripheral neuropathy did not demonstrate any abnormality of CSF Mg and PO4. In the multiple sclerosis group individual patients had elevated CSF concentrations of PO4 but the group as a whole is not different from the controls.

Hormones, electrolytes, and cerebrospinal fluid proteins in manic-melancholic patients.

The concentrations of insulin and thyroid hormones, tryptophan, electrolytes, urea, plasma proteins in the cerebrospinal fluid, and glucose in blood and cerebrospinal fluid in manic-melancholic patients were studied. As control groups served patients suffering from other psychiatric disorders as well as neurological and orthopedic patients. Apart from the blood values of thyroid hormones, the results showed no differences between the various diagnostic groups, neither in the abnormal states nor when recovered. For blood thyroxine and free thyroxine index, a statistically significant differences was seen in unipolar (melancholic) patients, namely a decrease concomitant with the clinical improvement. A tendency in the opposite direction of the thyroxine values was found in bipolar (melancholic) patients. In the manic group a marked decrease in the thyroxine values was obtained in the lithium-treated patients.

Composition of fluid from the notochordal canal of the coelacanth, Latimeria chalumnae.

Fluid from the notochordal canal of the coelacanth, Latimeria chalumnae, was analyzed for major inorganic and organic constituents and compared with blood serum from the same fish. Significantly or suggestively lower levels of sodium, magnesium, calcium, bicarbonate, sulfate, total carbohydrates, glucose, lactate, cholesterol, bound phosphate and total proteins were found in notochordal fluid than in serum, whereas potassium, chloride, urea, trimethylamine oxide, and total free amino acids were higher and inorganic phosphorus essentially identical. Osmolarity of notochordal fluid (1058 mOsm) exceeds that of serum (942 mOsm). A whitish precipitate in the fluid consisted of a matrix of fibers 100 A in diameter and of indefinite length. It resembled a sialoglycoprotein in composition and was stabilized by disulfide bonds. The fluid contained cellular debris.