Absorption of chlorophyll phytol in normal man and in patients with Refsum's disease

This study was made to determine the extent of absorption of chlorophyll phytol from the intestine of man, and the importance of chlorophyll as a source of the phytanic acid that accumulates in Refsum's disease. Uniformly (14)C-labeled pheophytin a (the Mg-free derivative of chlorophyll a) was fed to normal human subjects and to patients with Refsum's disease. Feces were collected and analyzed. In all subjects, 90-95% of the administered radioactivity was recovered in the feces, still largely in the form of pheophytin a. The phytol radioactivity recovered in the feces averaged about 95% of that in the administered material, which indicates that there had been little absorption of the phytol moiety. Similarly, after 250 g of cooked spinach had been fed to a normal subject, almost the entire phytol content was found in the feces. Less than 5% of the ingested spinach phytol was accounted for in the thoracic duct lymph of another subject. It was concluded that not more than about 5% of the ingested chlorophyll phytol is absorbed by man, whether normal or afflicted with Refsum's disease. On this basis we conclude that the major portion of the phytanic acid that accumulates in Refsum's disease could not be derived from dietary chlorophyll.     

Metabolism of phytol-U-14C and phytanic acid-U-14C in the rat

The metabolism of uniformly-labeled (14)C-phytol, (14)C-phytenic acid, and (14)C-phytanic acid was studied in the rat. Conversion of both phytol and phytenic acid to phytanic acid was demonstrated. Tracer doses of phytol-U-(14)C given orally were well absorbed (30-66%), and approximately 30% of the absorbed dose was converted to (14)CO(2) in 18 hr. After intravenous injection, 20% appeared in (14)CO(2) in 4 hr. Phytanic acid-U-(14)C given intravenously was oxidized at a comparable rate (22-37% in 4 hr) and was as rapidly oxidized as palmitic acid-1-(14)C (21% in 4 hr). Metabolism of these substrates was also studied in rats previously maintained on a diet containing 5% phytol by weight, which causes accumulation of phytanic acid, phytenic acid, and, to a lesser extent, phytol in blood and tissues. Despite the large body pools of preformed, unlabeled substrate in these animals, the fraction of an administered dose of phytol-U-(14)C or phytanic acid-U-(14)C converted to (14)CO(2) was not significantly diminished. These studies indicate that the rat has an appreciable capacity to degrade the highly branched carbon skeleton of phytol and its derivatives. Twenty-four hours after administration of phytol-U-(14)C, the lipid radioactivity remaining in the body was widely distributed among the tissues, highest concentrations being found in liver and adipose tissue. Four hours after intravenous administration of phytanic acid-U-(14)C, all of the major lipid classes in the liver contained radioactivity, most in triglycerides and phospholipids and least in cholesterol esters and lower glycerides. There was no demonstrable incorporation of mevalonate-2-(14)C or acetate-1-(14)C into liver phytanic acid when they were given intravenously to a rat previously fed phytol. Endogenous biosynthesis, if it occurs at all, must be extremely limited.     

Chlorophyll turnover in etiolated greening barley transferred to darkness: Isotopic (1-14C glutamic acid) evidence of dark chlorophyll synthesis in the absence of chlorophyll accumulation

Synthesis of chlorophyll was initiated in 5- to 6-day-old dark-grown barley (Hordeum vulgare L. cv. Clipper)seedlings by exposing them to light in the presence of 1-¹⁴ C glutamic acid supplied via the roots.The plants were then returned to darkness. At the end of light treatment (_T) and after 7 or 18 h dark treatment chlorophylls a and b were extracted, quantified (μgleaf¹). purified by HPLC to their magnesium-free derivatives (pheophytin a and b) and their molar radioactivities determined. After 2 h exposure to light followed by 6 h illumination in the presence of 1-¹⁴ C glutamic acid, seedlings had accumulated 4-7 nmol chlorophyll leaf¹ and had incorporated between 900-1 350 Bq (g fresh weight)¹ of radioactive label into the chlorophyll pool. When seedlings were transferred to darkness, label continued to be incorporated and after 18 h the radioactivity of the chlorophyll pool had increased by 300-700 Bq (g fresh weight)¹. Net chlorophyll content, however, remained constant during dark treatment. The increase in radioactivity of the chlorophyll pool in darkness represented the difference between a net increase of label incorporated into chlorophyll a and a small loss of label from chlorophyll b. The absence of measurable radioactivity in the phytol moiety of labelled chlorophyll a, extracted at the endof dark treatment, demonstrated thatincorporation of label was into the tetrapyrrole moiely of chlorophyll and not into the phytol chain. Light-independent incorporation of 1-¹⁴ C glutamic acid into chlorophyll of greening barley seedlings transferred to darkness indicates that chlorophyll synthesis continues when light is withheld. We interpret the net gain in radioactivity of chlorophyll in darkness, in the absence of a net gain in chlorophyll content, to chlorophyll turnover i.e. to simultaneous synthesis and breakdown of chlorophyll when etiolated greening barley seedlings are transferred to darkness.     

Urinary metabolites of cis-9,10-methylene octadecanoic acid. cis-3,4-Methylene hexanedioic acid and related compounds.

cis-3,4-Methylene hexanedioic acid has been discovered in human urine. It has been isolated and identified by mass spectrometry and synthesis. The daily excretion in nine subjects on a free diet was 88 mumol/day (range, 32 to 144 mumol/day). cis-3,4-Methylene hexanedioic acid was given orally to a rat. About 90% of the dose was recovered unchanged in the urine within 24 h. Intragastric administration of cis-9,10-methylene [9,10-3H2]octadecanoic acid to rats gave four labeled urinary metabolites. The major one was cis-3,4-methylene hexanedioic acid, the others were 2,3-methylene pentanedioic acid and isomers of methylene heptanedioic acid and methylene octanedioic acid. Within 72 h, about 40% of the administered radioactivity could be recovered from the urine and another 40% from the carcass. About 20% of the recovered radioactivity was found to be water. Of the radioactivity administered to rats orally as cis-9,10-methylene [9,10-3H2]octadecanoic acid methyl ester, about 50% could be recovered from the lymph of the thoracic duct within 9 h. Intraperitoneal administration of cis-9,10-methylene octodecanoic acid methyl ester to rats gave the same metabolites. Of the given amount, 50 mol % could be recovered from the urine as cis-3,4-methylene hexanedioic acid and 19 mol % as homologues within 38 days.     

The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.

The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.     

Stoichiometric determination of pheophytin in photosystem II of oxygenic photosynthesis

Pheophytin and chlorophyll extracted from oxygen-evolving photosystem II particles, chloroplast thylakoids and cyanobacterial cells were separated by column chromatography with DEAE-Toyopearl, and quantitatively determined by spectrophotometry. The molecular ratio of chlorophyll a+b to pheophytin a was about 100 in spinach photosystem II particles and about 140 in spinach thylakoids. Using flash spectrophotometry of P680 and measurement of flash-induced oxygen yield, the molecular ratio of the chlorophyll to the photochemical reaction center II was determined to be about 200 in the photosystem II particles. These findings suggest that the stoichiometry in photosystem II particles is one reaction center II and two pheophytin a molecules per about 200 chlorophyll molecules. The same stoichiometry for pheophytin to the reaction center II was obtained in the cyanobacteria, Anacystis nidulans and Synechocystis PCC 6714. A quantitative determination of pheophytin a and the electron donor P700 in stroma thylakoids from pokeweed suggests that photosystem I does not contain pheophytin.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

THE TEMPO OF LYMPHOCYTE RECIRCULATION FROM BLOOD TO LYMPH IN THE RAT

Radioactively labelled thoracic duct lymphocytes were obtained either by incubation in vitro with ³H-uridine or ¹⁴C-uridine or by giving potential donors repeated injections of ³H-thymidine finishing 17 days before thoracic duct cannulation. These labelled TDL were injected i.v. into syngeneic recipients which had been subjected to splenectomy and thoracic duct cannulation on the previous day. The tempo of lymphocyte recirculation from blood to lymph was reflected by the time at which radioactivity was recovered in the thoracic duct lymphocyte output of the recipient. This was measured by scintillation counting of 2-hourly fractional collections for 36 hr after the injection. Two lines of evidence showed that the majority of small lymphocytes which label intensely with radioactive uridine in vitro were uniform in their 'migration potential'with a modal blood to lymph transit time of 14–18 hr. By contrast the cells which were labelled in vivo with ³H-thymidine included a slower population with a modal transit time of 24–28 hr. These conclusions can be more fully interpreted in the light of recent evidence on thymic-independent ('B') lymphocytes.     

Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat

Tetrahydrolipstatin (THL) derived by hydrogenation from lipstatin, a lipase inhibitor produced by Streptomyces toxytricini, has been shown to inhibit in vitro the activity of all three lipases secreted to the gastro-intestinal tract; gastric lipase, pancreatic lipase and carboxylester lipase (cholesterol ester hydrolase). The effects of THL on intestinal absorption of fat (transport to the thoracic duct chyle) has now been investigated after intraduodenal infusion in a rat model. Absorption of label from oleic acid when administered with monoolein in micellar bile salt solution was not affected by THL in concentrations up to 10(-4) M calculated on the volume of the aqueous phase. Absorption of free cholesterol in micellar bile salt solution of the lipolytic products of triolein; oleic acid and monoolein, is not significantly affected at a concentration of THL of 10(-4) M. Absorption of cholesterol from cholesteryl oleate under the same conditions is almost completely inhibited. The results indicate that absorption of free cholesterol is not dependent on the activity of pancreatic cholesterol ester hydrolase. The absorption of emulsified triolein was not significantly affected by 10(-5) M THL but decreased to around 30% of the controls by a concentration 10-times higher. There was no significant decrease of cholesterol absorption when administered in emulsified triolein while absorption of cholesteryl oleate was reduced at both concentrations of THL and almost completely at 10(-4) M. Radioactivity from [2-14C]THL when administered emulsified in triolein was recovered in urine, bile and thoracic duct lymph to 10-14, 8-13 and 1-3%, respectively, largely independent on dose administered. Label from [1"-14C] THL was recovered in the same amounts in lymph but much less in bile and urine indicating that the amino acid moiety has been split off early in the absorption process.     

Cholesterol absorption and transport in thoracic duct lymph lipoproteins of nonhuman primates. Effect of dietary cholesterol level

The effect of dietary cholesterol level on cholesterol absorption and its subsequent transport in thoracic duct lymph lipoproteins was studied in two species of nonhuman primates, namely the African green monkey (Cercopithecus aethiops) and the cynomolgus macaque (Macaca fascicularis). Each animal served as its own control because each received sequential, intraduodenal infusions of two fat-rich liquid diets that differed only in the amounts of cholesterol. The percentage of dietary cholesterol absorbed was calculated by dividing the rate of appearance of exogenous cholesterol radioactivity in lymph by the rate of infusion of dietary cholesterol radioactivity at a time when lymph cholesterol specific activity was constant relative to that of diet. The percentage of dietary cholesterol absorbed was similar during both diet infusions in African green monkeys but was significantly decreased during the high cholesterol diet infusion in cynomolgus macaques. Rates of appearance of lymph total cholesterol mass were significantly increased during infusion of high cholesterol diets due to a statistically significant increase in cholesteryl ester transport rates. This increase was due in large part to the preferential esterification of exogenous cholesterol that was incorporated into lymph chylomicrons and VLDL. The rate of appearance in lymph of exogenous cholesterol significantly increased during the high cholesterol diet infusion while that of endogenous cholesterol decreased. This decrease or compensation in endogenous cholesterol transport occurred during absorption of increased levels of dietary cholesterol and apparently was due to an approximate 50% decrease in the absorption of lumenal cholesterol. Our data demonstrated that there was not a simple one-to-one relationship between the amount of isotopic dietary cholesterol absorbed from the intestinal lumen and the mass of cholesterol moved into lymph via the intestine. Rather, the amount of sterol transported into the body depends on the degree of the decrease in endogenous cholesterol transport and probably on the efficiency of cholesterol esterification during absorption.     

Green tea extract markedly lowers the lymphatic absorption and increases the biliary secretion of 14C-benzo[a]pyrene in rats

Previously, we have shown that green tea extract (GTE) lowers the intestinal absorption of lipids and lipophilic compounds in rats. This study was conducted to investigate whether GTE inhibits the intestinal absorption and biliary secretion of benzo[a]pyrene (BaP), an extremely lipophilic potent carcinogen, present in foods as a contaminant. Male rats with lymph or bile duct cannula were infused at 3.0 ml/h for 8 h via a duodenal catheter with lipid emulsion containing (14)C-BaP with or without GTE in PBS buffer. Lymph and bile were collected hourly for 8 h. The (14)C-radioactivities in lymph, bile and intestine were determined and expressed as % dose infused. Results showed that GTE drastically lowered the lymphatic absorption of (14)C-BaP (7.6±3.2% in GTE-infused vs. 14.4±2.7% dose/8 h in control rats), with a significantly higher amount of (14)C-radioactivity present in the small intestinal lumen and cecum in rats infused with GTE. GTE also markedly increased the hourly rate (3.9±0.1% dose/h in GTE-infused vs. 3.0±0.1% dose/h in control rats) and the total biliary secretion of (14)C-BaP (31.5±0.8% dose/8 h in GTE-infused vs. 24.3±0.4% dose/8 h in control rats). The findings provide first direct evidence that GTE has a profound inhibitory effect on the intestinal absorption of BaP and promotes the excretion of absorbed BaP via the biliary route. Further studies are warranted to investigate whether green tea could be recommended as a dietary means of ameliorating the toxicity and carcinogenic effect of BaP.     

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

Metabolism of Cyanox®, O,O-Dimethyl O-(4-Cyanophenyl) Phosphorothioate in the Rats

Orally administered Cyanox, O, O-dimethyl O-(4-cyanophenyl) phosphorothioate, labelled with carbon-14 at p-cyano group was easily absorbed from the gastrointestinal tract of male Wistar rats, and distributed into tissues. Elimination of the radioactivity was rapid and essentially complete; namely during 96 hr approximately 90.7% and 10% of total radioactivity were excreted respectively into urine and feces. Carbon-14 in expiration was negligible. No detectable amount of Cyanox was found in the urine and 0.01% of the administered compound was present in the feces. Degradation products in the urine were identified as demethylcyanox, demethylcyanoxon, p-cyanophenol and p-cyanophenylsulfate.     

Dopamine β-hydroxylase: Determination of half-life and appearance in lymph fluid after IV injection of 125I-DBH into rats

A 4 day half-life of dopamine beta-hydroxylase (DBH) was determined for rats injected IV with ¹²⁵I-rat DBH from the slow exponential component of radioactivity appearing in plasma, urine, feces and combined urine and feces. Half-life estimates for ¹²⁵I-rat DBH injected IV into WKY and SHR animals did not differ from Sprague Dawley (Zivic Miller) rats. Radioactivity declined in parallel in plasma, urine and feces following IV ¹²⁵I-rat DBH administration and each radioactivity falloff curve could be resolved into two components. The slow phase of the decline of radioactivity excreted into urine and feces from which DBH half-life was calculated occurred between 5 and 25 days after ¹²⁵I-rat DBH injection. The early fast phase which is associated with distribution of the exogenous protein in body fluids and tissues continued for approximately the first 140 hr after DBH injection. The distribution characteristics of IV administered active bovine DBH and ¹²⁵I-rat DBH into the lymphatic system were examined. After active bovine DBH or ¹²⁵I-rat DBH was injected IV into rats, active DBH or radioactivity, respectively, appeared in lymph fluid (thoracic duct) within 20 min; reached peak concentrations within 90 min, and thereafter, declined in parallel with the plasma concentration. The concentration of radioactivity in plasma and lymph fluid were found to be unequal at 9 hr but were equivalent 68–75 hrs after IV injection of ¹²⁵I-rat DBH. Based on the amount of active DBH or radioactivity which accumulates in lymph fluid it is clear that'a substantial amount (> 50%) of the DBH in blood circulates through the lymphatic channels. Analysis of parallel experiments with labelled serum albumin indicate that use of these methods to study plasma proteins do provide sensitive measures of biological half-life and lymphatic distribution characteristics. Specifically for DBH, the results of our study suggest that DBH normally circulates in plasma and lymph fluid with a biological half-life of 4 days.     

Natural killer cell activity in the rat. V. The circulation patterns and tissue localization of peripheral blood large granular lymphocytes (LGL)

Large granular lymphocytes (LGL) and T cells were separated from blood by centrifugation on discontinuous gradients of Percoll, were labeled with [3H]uridine or [111In]oxine, and were injected i.v. into syngeneic euthymic or athymic nude rats. The tissue distribution of these labeled cells was monitored for up to 24 hr after transfer by scintillation counting of tissue homogenates and autoradiography of tissue sections. In normal euthymic rats, the main sites of LGL localization were the alveolar walls of the lungs and spleen red pulp; however, they were not detectable in the major traffic areas of T lymphocyte recirculation, the spleen white pulp, and lymph nodes. Furthermore, the density of labeled LGL was very low in the small intestine, thymus, kidney, and liver, although on a per-organ basis, about 10% of the injected radioactivity was found in the liver by 24 hr post-injection. When 111In-labeled LGL were injected i.v. into rats with an indwelling thoracic duct cannula, they completely failed to enter the thoracic duct lymphocyte (TDL) population over an observation period of 6 days. This finding was markedly different from the results obtained with T cells and was consistent with the lack of natural killer and antibody-dependent cellular cytotoxicity activity observed among TDL, even in rats pretreated with the biological response modifier, poly I:C. LGL in athymic nude rats also failed to recirculate between blood and lymph. However, in contrast to normal euthymic animals, a significant increase in the localization of radiolabeled LGL to lymph nodes was observed in nude rats between 30 min and 24 hr. Taken as a whole, these findings define the areas within the lungs and spleen in which blood LGL normally localize, and clearly demonstrate that LGL do not normally recirculate between blood and lymph.     

The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat

A luminal supply of biliary phosphatidylcholine is important in the translocation of absorbed fat into lymph and in the amount and composition of phosphatidylcholine concurrently synthesized. This study was undertaken to determine whether the effect was due to absorbed lysophosphatidylcholine, to a specific (1-palmitoyl) biliary lysophosphatidylcholine or to extra choline supplied by lysophosphatidylcholine. Rats with bile fistulae and thoracic duct lymph fistulae were given test meals of oleic acid and monoolein (molar ratio 2 : 1) infused duodenally for 8 h. Addition of choline chloride to the test meal increased lymphatic output of triglyceride and phospholipid but not to values found previously in rats with supplements of bile phosphatidylcholine or with bile ducts intact. Addition of dioleoyl phosphatidylcholine increased triglyceride and phospholipid output to values found in rats with intact bile ducts. Since dioleoyl phosphatidylcholine was as efficient as biliary phosphatidylcholine it was concluded that a luminal supply of 1-palmitoyl lysophosphatidylcholine was not essential. It seemed likely from the smaller effect of supplemented choline and from the fatty acid composition of lymph phosphatidylcholine that the essential requirement was a supply of absorbed lysophosphatidylcholine for rapid reacylation to phosphatidylcholine.     

Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium

The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [¹⁴C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride-treated plants did not differ significantly in the amount of [¹⁴C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [¹⁴C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable ¹⁴C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated ¹⁴C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments.     

Trichinella spiralis: newborn larval migration route in rats reexamined

The route by which Trichinella spiralis newborn larvae migrate from the small intestine to striated muscle was studied in inbred AO and random-bred Sprague-Dawley rats. Newborn larvae were quantitatively recovered from the thoracic duct lymph, peritoneal cavity, and hepatic portal vein blood during the course of a primary infection with 4000 muscle larvae. The total recovery of newborn larvae assessed in this manner was compared with the number of muscle larvae in control rats receiving the same infection. In both strains of rats, most of the newborn larvae were recovered from hepatic portal vein blood, fewer than 3% of newborn larvae were recovered from the thoracic duct lymph and peritoneal cavity combined. Long-term drainage of thoracic duct lymph (greater than 24 hr) significantly increased newborn larval recovery over short-term drainage (less than 24 hr). We conclude that there are several natural pathways of newborn larval migration that result in muscle larval establishment. These include direct invasion of capillaries and lymphatics in the intestine as well as migration through the intestinal serosa to the peritoneal cavity. In both AO and Sprague-Dawley rats, greater than or equal to 97% of newborn larvae migrate via the hepatic portal vein blood to the general circulation.     

Changes in thoracic and right duct lymph flow and enzyme content during skeletal muscle stimulation.

In the pentobarbital-anaesthetized dog the effect of electrical stimulation of hindlimb skeletal muscles on thoracic and right duct lymph flow and enzyme content was examined. Increase in plasma creatine kinase (CK), L-aspartate-aminotransferase (AST) and lactic dehydrogenase (LDH) during 30-min muscle stimulation were not significantly altered by draining lymph. Both right duct and thoracic duct lymph flow trebled during stimulation. At the same time, the activity of the three enzymes examined decreased in right duct lymph and increased in thoracic duct lymph. Of the latter, only the increase in lymph CK was of a sufficient magnitude to have resulted in a detectable increase in plasma CK. CK was the smallest of the three enzymes studied and apparently preferentially entered the lymph, suggesting that the larger AST and LDH molecules were not likely to have entered the blood plasma directly from skeletal muscle. Rather their entry from some other tissue, possibly the formed elements of the blood, is indicated.     

A TLC-GLC procedure for the quantitation of algal photosynthetic pigments and their degradation products

A procedure employing and GLC techniques for the analysis of algal chlorophylls and their degradation products has been developed and evaluated. Algal pigments were separated on MN 300 cellulose plates developed in an ascending solvent system of hexane saturated with acetonitrile and n-propanol (100:0,4, v:v). Quantitation of chlorophyll a, b and pheophytin a, b were accomplished by GLC analysis of their phytol, following alkaline methanolic hydrolysis of the individual pigments. The amount of chlorophyllides/pheophorbides in a sample was estimated by its free phytol content. This technique is especially valuable for the evaluation of the pigment contents of near sediment phytoplankton and periphyton samples, where large quantities of chlorophyll degradation products and/or carotenoid pigments are generally present which may interfere significantly with the routine analytical methods.     

Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 +/- 0.3 chlorophyll (Chl) a and 1.8 +/- 0.1 beta-carotene; one cytochrome b(559) was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and beta-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.