Accumulation and metabolism of pipecolic acid in the brain and other organs of the mouse

The long-term accumulation of pipecolic acid, as well as its disappearance following exogenous administration was studied in brain and other organs of the mouse. Mice were pulse-injected intraperitoneally or intravenously with 1Ci[³H]D,l-pipecolic acid (6.9 nmol/mouse=2.9 g/kg). The total radioactivity retained in tissues was measured in brain, liver, and kidney, as well as in plasma during the period 1 min to 24 hr. TLC separation of DNP-derivatives was performed. Three features of the pattern of retention of pipecolic acid are most salient; first the rapid accumulation in brain, second the rapid secretion of this compound in the urine, and third the long-lasting steady levels of radioactivity maintained in brain.Sixty minutes after i.v. injection, the brain/plasma ratio is approximately 0.2 and approaches unity only at 5 hr. Following intraperitoneal injection the percent recovered as pipecolic acid in brain is 78% at 30 min and 71% at 120 min, suggesting a slow metabolic activity. Liver shows a different trend than brain with a slower accumulation and a faster disappearance. Kidney shows a pattern similar to plasma with a rapid secretion of radioactivity into urine which correlates well with the exponential decrease in plasma and urine. The administration of probenecid significantly increases radioactivity due to pipecolic acid in brain, liver, and urine. Formation of -aminoadipic acid, a known metabolite of pipecolic acid, can be demonstrated in kidney 30 min after intraperitoneal injection. The present data together with results obtained previously with intracarotid injections suggest that pipecolic acid is taken up in the mouse brain from the circulation. Most of the pipecolic acid taken up is rapidly removed through the circulation and secreted in the urine; however, a small part is retained and probably metabolized by brain and kidney.     

The conversion of lysine into piperidine,cadaverine, and pipecolic acid in the brain and other organs of the mouse

The biosynthesis of piperidine, a possible neuromodulator, and its presumed precursors cadaverine and pipecolic acid, has been investigated in the mouse under in vitro conditions. Conversion of lysine into piperidine was observed only in the intestines and is probably caused by the intestinal flora. Formation of cadaverine and pipecolic acid from lysine was observed in the brain, liver, kidney, and large intestine. In addition, pipecolic acid was formed in the heart. The possible contributions of the diet and of the intestinal bacteria to the endogenous pool(s) of piperidine are discussed.     

Lysine biosynthesis in Rhodotorula glutinis: properties of pipecolic acid oxidase.

Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to alpha-aminoadipic-delta-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 X 10(-3) M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.     

Accumulation,elimination, release and metabolism of pipecolic acid in the mouse brain following intraventricular injection

Following i.c.v. (intracerebral ventricular) injections ofd,l-[³H]pipecolic acid (PA), it is reabsorbed from the ventricles and redistributed to various brain regions. The highest accumulation is found in three brain regions ipsilateral to the injection site, hippocampus, neocortex, striatum, and in the diencephalon. Following preloading in vivo, the radioactivity is released from hippocampus slices in the perfusion medium after depolarization induced by high K⁺. During perfusion with a Ca⁺⁺ free medium containing EGTA, a significant reduction of release is observed.The radioactivity ofd,l-[³H]PA in the brain shows a more rapid phase of decrease from 0 to 2 hours and a slower phase from 2 to 5 hours. At 5 hours, only 28% radioactivity, represented mainly by PA, is left in the brain. Kidney secretion represents the major route of elimination of the injected PA. The presence of -aminoadipic acid both in brain and urine was observed. Probenecid (200 mg/kg) significantly increases the accumulation of i.c.v. injectedd,l-[³H]PA in brain and kidney. The presence of a regional accumulation of PA in certain brain regions, its metabolism in brain, its enhanced retention following probenecid administration and its Ca⁺⁺ dependent release following high K⁺ stimulation, all constitute indirect evidence for a neuronal localization of this brain endogenous iminoacid.     

Transport of pipecolic acid in adult and developing mouse brain

In an effort to develop an animal model of hyperpipecolatemia, the uptake of pipecolic acid (PA) in the brain and changes of PA levels in serum following administration ofd,l-PA were studied in the mouse using a new sensitive HPLC-EC method. Following i.p. injections (250 mg/kg) to adult male mice, the brain concentration peaks at 5–10 min (40 nmol/g). The level remains relatively stable up to 5 hrs and then declines slowly to 24 hrs. In serum, the level of PA increases rapidly to reach the maximum value at 10 min and then decreases rapidly in the first hour and continues to decline more slowly to 24 hrs. The net uptake of PA following administration of various amounts ofd,l-PA is saturable at low doses (3.9–15.6 mg/kg), and it increases linearly at higher doses in a dose-dependent manner up to the maximum dose (500 mg/kg) used in the present study. Kinetic analysis suggests the presence of two kinds of transport systems. These findings are in good agreement with the previous results usingd,l-[³H]PA in the mouse (7) andl-[¹⁴C]PA in the rat (13). There were no significant differences between uptake ofd-pipecolic acid andl-pipecolic acid (250 mg/kg, i. p., 10 min), suggesting the absence of stereospecificity for PA uptake in the mouse brain. Developmental changes in net brain uptake of PA following injections ofd,l-PA (250 mg/kg, s.c., 10 min) showed an age-dependent decrease which continues until adult levels are reached at four weeks after birth. The results suggest that the blood brain barrier (BBB) for PA is completed during the first month of life. Following administration ofd,l-PA (250 mg/kg, s.c.) to pregnant mice during the period 19–21 days of gestation, PA level increases in fetal brain to a maximum value at 2 hrs (420 nmol/g). This level is unchanged during 24 hrs. The maximum level of PA in fetal serum is reached at 30 min to 1 hr. The level gradually decreases after 1 hr over 24 hrs. These results indicate that PA taken up by the placenta and into the brain is transported from the fetal circulation. Our results also demonstrate that a higher amount of PA is taken up by the fetal than the adult brain. This finding is important in order to develop an animal model of hyperpipecolatemia in which high brain levels of PA should mimick those of human hyperpipecolatemic patients. Our results strongly support the hypothesis that high levels of PA present in brain during fetal life may exert a devastating effect on the development of the human CNS in hyperpipecolatemic children.     

Sulfur amino acid metabolism in the developing rhesus monkey brain: Interrelationship of taurine and glutamate

The relationship of taurine to glutamate, and to other amino acids, has been examined in the occipital lobe of the developing rhesus monkey. During development taurine decreases in concentration (4.96 mol/g in fetus to 1.52 mol/g in adult) while glutamate increases (7.92 mol/g in fetus to 11.26 mol/g in adult). When the concentration of taurine is plotted against that of glutamate in fetal, neonatal and adult animals there is a significant correlation in the fetal (p<0.01) and adult (p<0.01) but not in the neonatal occipital lobe samples. This correlation in both fetal and adult brain is specific for these two amino acids. Subcellular fractionation studies further indicate that this relationship may be of special importance in nerve endings.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Identification and characterization of pipecolic acid binding sites in mouse brain

Pipecolic acid (PA, piperidine-2-carboxylic acid) is the major product of lysine metabolism in the mammalian brain (Giacobini et al., 1980). In this study we have characterized the binding of [³H]PA to P₂ fraction membranes and its distribution in the mouse brain. The binding was found to be saturable (70 nM), temperature and Na⁺ and Cl^– dependent. A high affinity binding site with an apparentK _D of 33.2 nM and aB _max of 0.2 pmol/mg protein was demonstrated. The regional distribution of [³H]PA specific binding in mouse brain showed the highest concentration in cerebral cortex, thalamus and olfactory bulb. Unlabeled PA (10^–3–10^–11M) displaced specific binding of [³H]PA in a concentration dependent manner. Out of several substances tested, only proline showed a similar pattern of displacement. Pre-incubation of the membrane preparation with GABA (10^–3–10^–11M) resulted in either an increase or decrease of [³H]PA binding depending on the concentrations of GABA and PA. These results suggest a modulatory action of GABA on PA binding sites. The postnatal development of [³H]PA specific binding was studied in the whole brain of the mouse. [³H]Pipecolic acid binding increased progressively (8-fold) from one day after birth to 16 days. Following this developmental peak, the binding decreased gradually to 30 days at which age, adult values were attained.     

Quantitative determination and regional distribution of pipecolic acid in rodent brain

A rapid and sensitive method for the quantitative determination of pipecolic acid (PA), one of the three cyclic secondary imino acids present in mammalian brain is described. The quantification and identification of PA are accomplished in rat and mouse brain using high performance liquid chromatography with electrochemical detection (LCEC) and nipecotic acid (NPA) as an internal standard. The cyclic imino acids are derivatized with 2,4-dinitrofluorobenzene (DNFB) to dinitrophenyl derivatives. The remaining time for LCEC analysis is less than 30 min and the limit of sensitivity is in the lower picomole range. The levels of PA found in rat and mouse brain are comparable to those reported using gas chromatography/mass spectrometry. The regional distribution of PA shows higher concentrations of PA in hypothalamus, pons-medulla oblongata and cerebellum. The present results demonstrate that LCEC is sensitive enough to determine endogenous levels of PA in mg amounts of rodent brain tissue. Due to its simplicity and rapidity, the technique represents an alternative to existing methods. This method can also be used for determination of PA in CSF, blood or urine of hyperipecolic patients.     

Uptake of piperidine and pipecolic acid by synaptosomes from mouse brain

Piperidine is actively transported into the synaptosomal fraction of adult mouse brain. The transport mechanism appears to be Na⁺ independent but is temperature dependent and sensitive to ouabain. Analysis of kinetic experiments indicates only a low-affinity transport system to be present. By contrast the uptake ofD,L-[³H]pipecolic acid at a concentration of 4×10^–7 M was temperature and Na⁺ dependent, ouabain sensitive, and revealed a two-component system with aK _m =3.9±0.17×10^–6 M,V _max=129±6 pmol/mg protein/3 min for the high-affinity system and aK _m =90.2±4.3×10^–6 M,V _max=2.45±0.19 nmol/mg protein/3 min for the low-affinity system. Compounds structurally related to pipecolic acid such as glycine,l-proline, 4-amino-n-butyric acid, and 5-amino-n-valeric acid showed an inhibitory effect on uptake at a concentration of 10^–4 M. The demonstration of biosynthesis of pipecolic acid in mouse brain and the presence of a high-affinity sodium-dependent uptake system suggest a physiological role of this substance in the central nervous system.     

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.     

Lysine metabolism in mammalian brain: an update on the importance of recent discoveries

The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ?¹-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, α-aminoadipate δ-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ?¹-piperideine-2-carboxylate (P2C) and its reduced metabolite l-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to l-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [μ-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3′-triiodothyronine (T₃) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Simultaneous analysis of pipecolic acid with proline in the brain by selected ion-monitoring technique

A method for the simultaneous analysis of pipecolic acid and proline in the brain is developed. The qualification and quantification of pipecolic acid and proline are accomplished with gas chromatography/mass spectrometry including a selected ion-monitoring technique by using deuterium-labeled proline as an internal standard, after the amino and carboxylic groups of these cyclic amino acids are derivatized with boron trifluoride methanol complex and heptafluorobutyric anhydride. The lower limit of quantification for the method is picomole levels and the concentration of pipecolic acid and proline in rat whole brain is determined to be 1.05 and 71.50 nmol/g of tissue, respectively.