Degradation of somatomedin A by various organ homogenates from rats

Degradative activities of somatomedin A (SMA) have been examined in various tissue homogenates of rat using trichloracetic acid precipitable radioactivity of 125I-SMA. Kidney and testis showed higher specific activities and liver and brain lower activities. They were dependent on SH reagents; 0.5 mM HgCl2 inhibited the degradative activity of liver completely and 1 mM dithiothreitol (DTT) augmented the activity slightly. The activities in liver were separated by differential centrifugation; about 90 per cent of the total activity in the whole homogenate was recovered in the supernatant fraction at 100,00 x g for 60 min, and 10 per cent in the precipitate. The pH profile of each fraction was different; that of the supernatant showed a single peak at pH 7.4 and that of the pellet revealed two peaks at pH 5.9 and 7.4. However, both fractions showed similar SH-dependency.     

Enzymatic oxidation of all-trans retinal to retinoic acid in rat tissues

The 100,000 x g supernatant (cytosolic) fraction of rat tissue homogenates catalyzes the oxidation of all-trans retinal to retinoic acid. Kidney, testis, and lung were the most active of the tissues examined. The presence of enzyme activity in liver and intestine could be detected only when a substrate concentration beyond the saturation point for retinal reductase was used. Spleen, brain, and plasma had no activity. Boiled supernatants did not catalyze the reaction. The enzymatic product was chemically and physically identified as retinoic acid. The cytosol of kidney tissue also catalyzed the conversion of retinol to retinoic acid. These data indicate that kidney tissue has the highest retinal oxidase activity and suggest that it may play a major role in the oxidative metabolism of retinol in the body.     

Effect of triiodothyronine on the content of phospholipids in the rat liver nuclei.

The aim of the present study was to examine the effect of triiodothyronine (T3) on the content of phospholipids and on the incorporation of blood-borne palmitic acid into the phospholipid moieties in the nuclei of the rat liver. T3 was administered daily for 7 days, 10 microg x 100 g(-1). The control rats were treated with saline. Each rat received 14C-palmitic acid, intravenously suspended in serum. 30 min after administration of the label, samples of the liver were taken. The nuclei were isolated in sucrose gradient. Phospholipids were extracted from the nuclei fraction and from the liver homogenate. They were separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. The content and radioactivity of each fraction was measured. It was found that treatment with T3 reduced the content of phosphatidylinositol and increased the content of cardiolipin in the nuclear fraction. In the liver homogenate, the content of phosphatidylinositol decreased and the content of phosphatidylethanolamine and cardiolipin increased after treatment with T3. The total content of phospholipids after treatment with T3 remained unchanged, both in the nuclear fraction and in the liver homogenate. T3 reduced the specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin and had no effect on the specific activity of sphingomyelin and phosphatidylinositol both in the fraction of the nuclei and the liver homogenate. It is concluded that excess of triiodothyronine affects the content of phospholipids in the nuclei. The changes in the content of phospholipids in the nuclei largely reflect changes in their content in the liver.     

Increased content of calmodulin in morris hepatoma 5123 t.c.(h)

The content of calmodulin in the 100,000 × g supernatant and particulate fractions in Morris hepatoma 5123 t.c.(h), assayed by its ability to activate the Ca²⁺-activatable cAMP phosphodiesterase, was significantly higher (about 44%) than that in normal or host liver. Only one peak of calmodulin activity was detected when 100,000 × g supernates (350 mg protein) from sonicated homogenates of normal liver and hepatoma were fractionated on a DEAE cellulose column, eluting at a sodium acetate concentration of 0.65 M. The total calmodulin activity eluted from the hepatoma supernatant was 240% higher than that from nornal liver. The increased content of calmodulin in the hepatoma may contribute to the state of abnormal cell proliferation in this tissue.     

Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans.

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-beta-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 x g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100,000 x g centrifugation. When both fractions (the 100,000 x g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100,000 x g supernatant. In addition, the 100,000 x g precipitate, which was prepared from the 110 x g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100,000 x g supernatant activates galactosyltransferase-I activity.     

Partial purification and characterization of insulin protease and its intracellular inhibitor from rat liver

Insulin protease was purified 700-fold from rat liver homogenate by combined ultracentrifugation, ammonium sulfate fractionation, and glucagon-Sepharose-4B affinity chromatography. Optimum degradation of insulin was observed at pH 7.6 with the purified protease whose Km was 24 nM. The enzyme activity was inhibited completely by N-ethylmaleimide, p-hydroxymercuribenzoate, and heavy metals at 1 mM, whereas at the same concentration glutathione and mercaptoethanol stimulated the protease activity. These results indicate that the catabolic activity of the protease is sulfhydryl dependent. Furthermore, the activity of insulin protease was also enhanced by calcium and other divalent metal ions at a concentration of 1 mM. When supernatants, recovered from rat liver homogenates after centrifugation at 100,000g, were subjected to combined Sepharose 4B-insulin protease affinity chromatography and dialysis, a potent inhibitor of insulin protease was obtained which was heat stable. On the basis of kinetic studies, the inhibition of insulin degradation caused by this inhibitor was of the competitive type. Greater than 90% of the inhibitor activity was retained on dialysis with tubing with an inclusion limit of 3500 Da, whereas only 10% of this activity could be retained in dialysis tubing with an exclusion limit of 15,000 Da. These findings suggest that the insulin protease inhibitor is a low-molecular-weight protein. Analysis of homogenates from 13 different tissues of the rat showed that the highest levels of insulin protease inhibitor activity were associated with those tissues which have the highest capacity to degrade insulin. These data suggest that insulin protease and insulin protease inhibitor may be an important natural regulatory mechanism of insulin activity.     

肝细胞生长因子基因修饰的人脐带间充质干细胞对大鼠慢性肝损伤的治疗研究

目的：研究肝细胞生长因子（HGF）基因修饰的人脐带间充质干细胞（MSC）对Wistar大鼠慢性肝损伤的治疗作用。方法：利用携带人HGF基因的重组腺病毒（Ad～HGF）对MSC进行基因修饰;通过皮下注射CCl4-橄榄油溶液建立大鼠肝损伤模型;56只Wistar雄性大鼠随机分为正常对照组、模型组、MSC组、HGF组和HGF／MSC组,分别尾静脉注射生理盐水、MSC、Ad-HGF或HGF／MSC,通过血清肝功能检测及肝组织的生化指标、病理切片等评价治疗效果。结果：利用腺病毒将HGF基因转入MSC并确定了最佳感染条件。CCl4-橄榄油诱导4周后,动物肝脏外观出现明显的脂肪变性和血清转氨酶明显升高,表明成功诱导了大鼠慢性肝损伤模型。经过4周治疗,治疗组动物的体重明显升高、肝指数明显降低,动物存活率高于模型组（模型组：50％;MSC组：70％;Ad-HGF组：70％;HGF／MSC组：90％）。肝功能指标测定结果显示,与模型组比较,HGF／MSC组动物的天门冬氨酸氨基转移酶（406.75±35.98 vs.513.75±12.71U／L,P〈0.01）、丙氨酸氨基转移酶（124.6±10.6 vs.169.67＋15.38 U／L,P〈0.05）和总胆红素（14.6±2.08 vs.19.25±1.38g／dL,P〈0.01）均明显降低,白蛋白含量（29.1±1.3vs.22.05±2.61g／L,P〈0.05）明显升高;Ad-HGF组只有天门冬氨酸氨基转移酶（436.0±18.40vs.513.75±12.71U／L,P〈0.05）明显降低;而单纯MSC治疗对肝功能改善不明显。丙二醛测定结果显示,治疗后动物肝组织中的过氧化反应均较模型组显著减弱。HGF／MSC组和MSC组动物肝组织中的羟脯氨酸含量与模型组比较明显降低（69.27±14.58,63.23±13.23 vs.96.59±15.05mg／mL,P〈0.01）。上述实验结果提示3种治疗方式对CCl4-橄榄油引起的大鼠肝损伤均有一定的治疗效果,可减轻CCl。导致的肝损伤程度,增加动物体重,降低肝脏指数,减轻肝组织的过氧化反应,提高动物的存活率,其中HGF／MSC治疗效果最为明显,而且细胞注射没有引起动物的不良反应。结论：3种治疗方式均可改善CCl。导致的肝损伤,其中以HGF／MSC对肝损伤的治疗效果最好。本实验为HGF／MSC的临床研究提供了新的实验数据,为治疗肝损伤提供了新的治疗措施。     

Intracellular free iron in liver tissue and liver homogenate: studies with electron paramagnetic resonance on the formation of paramagnetic complexes with desferal and nitric oxide.

Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.     

Quantitative determination of unchanged cisplatin in rat kidney and liver by high-performance liquid chromatography

A quantitative analytical method for measuring unchanged cisplatin (CDDP) and high- and low-molecular-mass metabolites (fixed and mobile metabolites) in rat kidney and liver was developed. Unchanged CDDP, separated from fixed and mobile metabolites in tissue homogenates by consecutive procedures of fractionation and ultrafiltration, was determined by high-performance liquid chromatography (HPLC) with post-column derivatization. Although unchanged CDDP was found to be partly metabolized to fixed metabolites during the preparation of cytosolic ultrafiltrates, the recovery of unchanged CDDP gave a constant value (about 70%), which was independent of tissue type and CDDP concentration (from 1 to 10 μg/ml). The detection limit for unchanged CDDP in the cytosolic ultrafiltrate was 20 ng/ml, corresponding to a concentration detection limit of 65 ng Pt per g of tissue in the kidney and liver. The concentrations of fixed and mobile metabolites were determined as platinum concentrations in the tissue homogenate and in the cytosolic ultrafiltrate using atomic absorption spectrometry after correcting for transformation of unchanged CDDP to fixed metabolites. The distribution of unchanged CDDP, mobile metabolites and fixed metabolites in rat kidney and liver, after bolus injection of CDDP (5 mg/kg), was determined using this method.     

Catechol-o-methyltransferase in rat erythrocyte and three other tissues: comparison of biochemical properties after removal of inhibitory calcium

The biochemical characteristics of soluble catechol-O-methyltransferase (COMT) activity in rat erythrocytes were compared with the properties of the soluble enzyme in rat liver, heart, and brain. COMT was measured by a procedure that avoided artifacts of some other assay procedures including inhibition of the enzyme by endogenous calcium. After the removal of calcium from the reaction mixture the apparent Michaelis-Menten constants for the two cosubstrates of the COMT reaction, S-adenosyl-1-methionine (SAM) and 3,4-dihydroxybenzoic acid (DBA), were similar in tissue preparations of rat liver, brain, heart and blood. The apparent Km values for the four tissues ranged from 5.7 to 6.7 x 10(-6) M and from 0.9-1.4 x 10(-4) M for SAM and DBA, respectively. The optimal pH and the optimal concentration of magnesium for the assay of red blood cell COMT were also similar to those for the enzyme in the three other rat tissues. After the removal of endogenous calcium, COMT activity in all four tissues was inhibited by the addition of calcium, and the [CaCl2] necessary to inhibit the enzyme activity 50% was 3-5 x 10(-4) M in all cases. The relative activities of COMT in the rat heart, brain, erythrocyte, and liver when expressed per g tissue or per ml of packed red blood cells were 1 to 1.15 to 1.58 to 140, respectively.     

Calcium administration increases calcium-binding protein regucalcin concentration in the liver of rats

The alteration of regucalcin concentrations in the liver and serum of rats administered orally calcium is investigated. Rats received a single oral administration of calcium chloride solution (25, 50 and 75 mg Ca/100 g body weight). The administration of calcium (50 mg/100 g) produced a significant increase in liver regucalcin concentration between 30 and 180 min after the administration, while serum regucalcin concentration was not altered appreciably. The effect of calcium administration increasing liver regucalcin concentration was also seen with the dose of 25 mg/100 g. When liver cytosol prepared from normal rats was incubated for 6 h in the presence of 10 M Ca²⁺, the cytosolic regucalcin concentration at 3 and 6 h of incubation was decreased about 20% (p<0.05) as compared with the value at zero time point, indicating that the presence of Ca²⁺ does not inhibit the decomposition of liver cytosolic regucalcin. Moreover, serum regucalcin concentration was not significantly altered by the incubation for 6 h at 37°C, indicating a stability of regucalcin in rat serum. This suggests that the calcium administration-induced in liver regucalcin concentration is not based on the inhibition of regucalcin release from liver to serum. The present study demonstrates that regucalcin in the liver is clearly increased by calcium administration, presumably due to stimulating the protein synthesis.     

Determination of AMP in the rat heart using skeletal muscle AMP deaminase

A new simple enzymatic method for measuring AMP content in freeze-clamped rat heart is presented. The method is based on the ammonia estimation after the deamination of 5'-AMP by muscle 5'-adenylic acid deaminase. The minimum detectable amount of AMP was about 1.5 nmol. The recovery of AMP added to the tissue homogenate was 94%. The variance coefficient evaluated by assaying five samples from one tissue extract was equal to 5%. AMP content of rat heart (0.28 mumol/g wet tissue) is comparable with the values reported by others.     

The metabolism of progesterone by animal tissues in vitro. Sex and species differences in conjugate formation during the metabolism of [4-14C]progesterone by liver homogenates

1. Sex and species differences during the metabolism of [4-¹⁴C]progesterone by liver homogenates from rat, rabbit, guinea pig and hamster have been investigated. 2. Liver homogenate from male rat formed `water-soluble' metabolites faster and in significantly larger amounts than did liver homogenate from female rat. About 65–70% of the added progesterone was conjugated as glucuronide by liver homogenate from male rat and about 45–50% by that from female rat. Liver homogenate from male rat also formed glucuronides faster than did liver homogenate from female rat. Sulphate formation was low (8–16%) in liver homogenates from both male and female rats. 3. Hamster-liver homogenate did not show any sex difference in the rate of formation of `water-soluble' metabolites, but a sex difference was observed in the amount of free steroids recovered at low tissue:steroid ratios. Liver homogenate from female hamster formed glucuronides faster and in significantly larger amounts than did liver homogenate from male hamster, the reverse of what was found in rat liver. 4. Liver homogenates from male and female rabbits and guinea pigs formed `water-soluble' metabolites that were almost entirely glucuronides. 5. Neither rabbit liver nor guinea-pig liver showed any significant sex difference in the rate or amount of formation of total `water-soluble' metabolites or glucuronides, but guinea-pig liver was considerably less active than rabbit liver. 6. Glucuronides were quantitatively the major type of conjugate formed by the liver homogenates from both sexes of all species except the male hamster.     

Time course of distribution to tissues of 125I-somatomedin A injected into the rat

125I-somatomedin A (SMA) was injected iv into rats. Distribution studies in rats showed concentrations of radioactivity to be high in kidney and plasma, low in brain, and intermediate in other tissues. The concentration of total and trichloracetic acid (TCA) precipitable radioactivity in rat blood and tissues fell at rapid rate. Ninety per cent of the radioactivity was in the urine in 24 hr, and only 15% of urine radioactivity was TCA precipitable. The half-life of the radioactivity in TCA-precipitable fraction from blood and that from tissues were nearly identical (about 6 hr). In both liver and kidney, TCA-precipitable radioactivity was detected in membrane and/or organellar fraction and cytosol fraction. Sephadex G-200 chromatography at neutral PHY AT NEUTRAL PH of plasma after injection of 125I-SMA revealed 3 peaks of radioactivity in higher molecular weight region than purified SMA.     

Separation, quantitation and distribution of dolichol and dolichyl phosphate in rat and human tissues

Two procedures for quantitative determination of dolichol were studied and these were applied to analyze tissue and subcellular distribution. In the first procedure the dolichols were oxidized with Cr2O3 and reduced with NaB3H4. The radioactivity in the individual dolichols was measured using reversed-phase thin-layer chromatography. In the second procedure, dolichols were analyzed by high-pressure liquid chromatography. For determination of dolichyl phosphates the lipid extract was subjected to acid and alkaline hydrolysis, and after hydrolysis with acid phosphatase the distribution was determined by high-pressure liquid chromatography. Recovery was monitored by the addition of dolichol D15 and D23 phosphate to the homogenate. Rat spleen had the highest dolichol content (114 micrograms/g) followed by lower content in rat liver and brain. The distribution pattern was similar in all organs, with 18 and 19 isoprene residues as dominating components. Human organs contain considerably higher concentrations of dolichol, with the 19 and 20 isoprene residues as the main components. In rat liver, outer mitochondrial and Golgi membranes, lysosomes and plasma membranes contain considerable amounts of dolichol. A drastic increase in dolichol content was observed in rat liver hyperplastic nodules while human liver cirrhosis and hepatocarcinoma showed a marked decrease in dolichol. In the latter case, the distribution pattern was also changed. Of the total amount of dolichol present in the tissues, 2% was phosphorylated in human liver, 10% in human testis and 18% in rat liver. In rat liver mitochondria and in microsomes 4 and 31%, respectively, of the polyprenols were in activated form. The results demonstrated that dolichyl phosphate and dolichol concentrations were regulated by different mechanisms and that the two forms possessed an independent distribution.     

Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial exposures to paraoxon or parathion before they become saturated, whereas A-esterases may contribute to paraoxon detoxication in repeated exposures to paraoxon or parathion because they will not become inhibited and will remain catalytically active unlike the carboxylesterases. The importance of carboxylesterases in detoxication of paraoxon was verified by an in vivo study. In rats pretreated with tri-o-tolyl phosphate, an in vivo carboxylesterase inhibitor, brain acetylcholinesterase was significantly inhibited after intravenous exposure to parathion. No significant inhibition of brain acetylcholinesterase was observed in rats pretreated with corn oil.     

Inhibition of cyclic nucleotide phosphodiesterase activity by an endogenous factor.

Cyclic nucleotide phosphodiesterase activity of several tissues of rat is inhibited by an endogenous factor isolated from rat adipocytes following exposure of these cells to agents that raise intracellular cyclic AMP levels. The inhibitory action was demonstrated with varying cAMP concentrations from 0.1-400 muM. Enzyme from 10,000 X g supernatant of epididymal adipose tissue was inhibited approximately 2-3 fold more than the plasma membrane of adipocytes by a given concentration of the feedback regulator. Kinetic analysis of cAMP phosphodiesterase of plasma membrane showed that feedback regulator (8.8 U/ml) inhibited the Vmax 48%. The maximum inhibition of phosphodiesterase by feedback regulator (20 U/ml) was about 80%. The apparent Km for cAMP was increased. The ability of phosphodiesterase from several tissues of rat (10,000 X g supernatant) to hydrolyze cAMP and cGMP was tested. Feedback regulator inhibited cGMP hydrolysis in cardiac muscle and 5 other tissues 23-92% more than it inhibited the hydrolysis of cAMP. The physiological significance of this inhibitory effect can begin to be clarified when the feedback regulator is purified to homogeneity and characterized.     

Studies on the binding of mercury in tissue homogenates

1. This paper describes an attempt to learn more about the binding of Hg(2+) to tissues at pharmacological concentrations of this metal. Other methods were not applicable to such low concentrations of mercury. 2. The method involved equilibrium dialysis of Hg(2+) against 1% homogenates of rat kidney or liver in the presence of penicillamine. Two classes of mercury-binding sites were observed, one class having a chemical affinity for mercury 100-fold greater than the other class. The binding capacities of the class of higher and lower affinity were respectively 1.0x10(-7) and 30x10(-7)mole of mercury/g. wet wt. of tissue. The same classes of binding sites were found in both liver and kidney homogenates. 3. The binding sites of both classes reacted with only one valency of Hg(2+), the other valency forming a bond with penicillamine. Thus the total binding capacities of both classes are equivalent to 50% of the total reactive protein-bound thiol groups in the homogenate. 4. The results eliminate three possible mechanisms for the preferential accumulation of mercury by kidney. They support the idea that the permeability changes in kidney cells resulting in diuresis are similar to the permeability changes produced on the membranes of other mammalian cell species by mercury.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of the lipophilic antipsychotic drug chlorpromazine in rat plasma and brain tissue

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for quantification of chlorpromazine in rat plasma and brain tissue. Chlorpromazine was extracted from rat plasma and brain homogenate using liquid-liquid extraction. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/20 mM ammonium formate (pH 4.25 adjusted with formic acid) with gradient elution. Chlorpromazine was detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. The LLOQ was 0.2 ng/ml for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/ml for plasma and from 0.833 to 833.3 ng/g for brain tissue. The correlation coefficient (R(2)) values were more than 0.998 for both plasma and brain homogenate. The precision and accuracy for intra-day and inter-day were better than 7.54%. The relative and absolute recovery was above 84.9% and matrix effects were lower than 5.6%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of chlorpromazine after chronic treatment.     

Ntau-methylhistidine content of mixed proteins in various rat tissues.

In order to use Ntau-methylhistidine (3-methylhistidine) excretion in the urine as a measure of muscle protein breakdown, it is necessary to demonstrate that other tissues are not important sources of this protein constituent. Accordingly, the concentration of Ntau-methylhistidine in blood serum and in the mixed proteins of heart, brain, lung, kidney, diaphragm, spleen, testis, stomach, liver and hind leg skeletal muscle was measured in male rats of approx. 400 g body weight. The free Ntau-methylhistidine concentration of rat serum was less than 2 nmol per ml. In contrast, measurable amounts of Ntau-methylhistidine were found in the mixed proteins of all tissues and organs examined. The highest concentration was found in skeletal muscle (658 nmol/g tissue). Assuming muscle mass to be 45% of body weight, it has been estimated that the muscle contains more than ten times the total amount of this amino acid present in all of the other organs analyzed, which together account for about 20% of total body weight. These findings indicate that skeletal muscle is likely to be the major source of urinary Ntau-methylhistidine and the latter is, in consequence, a reflection of myofibrillar protein breakdown in skeletal muscle.