Sensitive,selective gas chromatographic—mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol-producing activity

One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[¹³C]sorbitol, ¹³C₆H₁₄O₆, 98.7%) as an internal standard. Gas chromatography—mass spectrometry (GC—MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[¹³C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1–8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r=0.999, r=0.997 and r=0.997, respectively. The precision of the GC—MS measurement of standard solution was C.V.=4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, K_m and V_max values of the reductase in erythrocytes were 115±19 mmol/l and 4.42±0.26 nmol/min/g of hemoglobin. In human liver, on the other hand, they were 75±132 mmol/l and 0.77±0.090 nmol/min/mg of protein, respectively. This difference of K_m values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.     

Interaction between polyhydroxy compounds and vanadate ions: electrophoresis and composition of complexes

Several polyhydroxy compounds form anionic complexes with vanadate ions. The V/polyol ratio in several complexes has been determined. In the pH range 5–9.5, the complexing agent is likely to be a metavanadate ion, (VO)_n^n-. Electrophoresis in sodium metavanadate solution is particularly useful for the separation of hexitols, hex-2-uloses, and several disaccharides.     

The determination of inorganic orthophosphate in biological systems

A simple and rapid method is described for determining P_i by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 10³ at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other P_i assays. Linearity is observed in the range 0–1.2 μmoles P_i and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (ε_{M, 390nm} = 2.79 · 10³) but linearity is maintained up to 1 μmole P_i and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of P_i in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with ε_{M, 820nm} of 9.9 · 10³ at pH 4.0, and 1.8 · 10⁴ under more acidic conditions.     

A sensitive method for measuring pyrophosphate in the presence of a 10,000-fold excess of orthophosphate using inorganic pyrophosphatase

A simple method for measuring PP_i at concentrations down to 2 μm has been devised using the ability of inorganic pyrophosphatase to be inactivated by fluoride in the presence of PP_i. Orthophosphate (20 mm) and a number of other compounds did not interfere with the assay. The applicability of the method for direct measurement of PP_i in urine is demonstrated.     

Osmoregulatory Responses of Fungi Inhabiting Standing Litter of the Freshwater Emergent Macrophyte Juncus effusus

Standing litter of emergent macrophytes often forms a major portion of the detrital mass in wetland habitats. Microbial assemblages inhabiting this detritus must adapt physiologically to daily fluctuations in temperature and water availability. We examined the effects of various environmental conditions on the concentrations of osmoregulatory solutes (polyols and trehalose) and the respiratory activities of fungal assemblages inhabiting standing litter of the freshwater emergent macrophyte Juncus effusus. Under field conditions, the concentrations of osmolytes (polyols plus trehalose) in fungal decomposers were negatively correlated with plant litter water potentials (r = −0.75, P < 0.001) and rates of microbial respiration (r = −0.66, P < 0.001). The highest concentration of osmolytes (polyols plus trehalose) occurred in standing litter exposed to desiccating conditions (range from wet to dry, 0.06 to 0.68 μmol · mg of fungal biomass⁻¹). Similar fluctuations in polyol and trehalose concentrations were observed in standing litter wetted and dried under laboratory conditions and for four predominant fungal decomposers of J. effusus grown individually on sterilized Juncus leaves. These studies suggest that fungal inhabitants associated with standing litter of emergent macrophytes can adjust their intracellular solute concentrations in response to daily fluctuations in water availability.     

Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5–green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H⁺-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.     

14C-Assimilate pattern and kinetics of photosynthetic 14CO2-assimilation of the marine red alga Bostrychia scorpioides

Summary Occurrence and metabolism of dulcitol and sorbitol in the marine red alga Bostrychia scorpioides (Huds.) Mont. (Ceramiales: Rhodomelaceae) were investigated. Both hexitols are rapidly ¹⁴C-labelled during photosynthesis in a H¹⁴CO₃-seawater medium and are accumulated at comparable rates. The absolute quantity amounts to about 3.2% on a dry weight basis; the percentage of ¹⁴C-labelling after 60 min is 30% for dulcitol and 40% for sorbitol. Additionally small amounts of free [¹⁴C] glucose were found. Pulse labelling experiments and changes in specific activity provide evidence that both hexitols are rapidly available respiratory substrates, which, however, are probably not interconvertible with polymeric compounds. Some chemotaxonomic aspects are discussed.     

Cryoprotectant Biosynthesis and the Selective Accumulation of Threitol in the Freeze-tolerant Alaskan Beetle, Upis ceramboides

Adult Upis ceramboides do not survive freezing in the summer but tolerate freezing to −60 °C in midwinter. The accumulation of two cryoprotective polyols, sorbitol and threitol, is integral to the extraordinary cold-hardiness of this beetle. U. ceramboides are the only animals known to accumulate high concentrations of threitol; however, the biosynthetic pathway has not been studied. A series of ¹³C-labeled compounds was employed to investigate this biosynthetic pathway using ¹³C{¹H} NMR spectroscopy. In vivo metabolism of ¹³C-labeled glucose isotopomers demonstrates that C-3—C-6 of glucose become C-1—C-4 of threitol. This labeling pattern is expected for 4-carbon saccharides arising from the pentose phosphate pathway. In vitro experiments show that threitol is synthesized from erythrose 4-phosphate, a C₄ intermediate in the PPP. Erythrose 4-phosphate is epimerized and/or isomerized to threose 4-phosphate, which is subsequently reduced by a NADPH-dependent polyol dehydrogenase and dephosphorylated by a sugar phosphatase to form threitol. Threitol 4-phosphate appears to be the preferred substrate of the sugar phosphatase(s), promoting threitol synthesis over that of erythritol. In contrast, the NADPH-dependent polyol dehydrogenase exhibits broad substrate specificity. Efficient erythritol catabolism under conditions that promote threitol synthesis, coupled with preferential threitol biosynthesis, appear to be responsible for the accumulation of high concentrations of threitol (250 mm) without concomitant accumulation of erythritol.Cold-hardy terrestrial insects from temperate to arctic regions exhibit two physiological strategies to survive subzero temperatures: freeze tolerance and freeze avoidance (1–3). Freeze-tolerant organisms survive ice in their extracellular fluids but are generally susceptible to intracellular freezing (3). Freeze-avoiding insects do not survive freezing of their body fluids and typically supercool beyond the lowest environmental temperature they are likely to experience in nature (4).Both freeze-tolerant and freeze-avoiding insects commonly produce polyols, also known as alditols, to mitigate the effects of low temperature (5). Polyols are biocompatible solutes that lack toxicity, even at molar concentrations, and stabilize native protein structure at low temperature (6). However, the functions of polyols depend on the overwintering strategy. In freeze-tolerant organisms, polyols reduce the fraction of frozen water, thereby preventing excessive intracellular dehydration. In contrast, polyols promote supercooling in freeze-avoiding organisms (5). Glycerol is the most commonly accumulated polyol in both freeze-tolerant and freeze-avoiding insects. However, overwintering insects are taxonomically diverse and produce various polyols in response to low temperature, including ethylene glycol, erythritol, mannitol, ribitol, sorbitol, and threitol (6).The accumulation of sorbitol and threitol in the hemolymph of the freeze-tolerant beetle, Upis ceramboides, is associated with increasing cold tolerance (7). In the summer, when neither sorbitol nor threitol are detectable in the hemolymph, U. ceramboides do not survive freezing. As sorbitol and threitol accumulate in the hemolymph, there is a concomitant decrease in lethal temperature to −60 °C (8).Polyols are typically produced in the fat body by the actions of two enzymes, a NADPH-dependent polyol dehydrogenase and a sugar phosphatase, on a sugar phosphate (e.g. glucose 6-phosphate) in constitutive metabolic pathways such as glycolysis or the pentose phosphate pathway (PPP)² (9). However, threitol biosynthesis does not fit this pattern because T4P, the putative metabolic precursor of threitol, is not observed in normal constitutive metabolism. Threitol is a biologically rare compound, and U. ceramboides are the only organisms known to accumulate it at high concentrations (∼250 mm) (7, 10), although low concentrations (≤2 μg/mg fresh mass or ∼3 mm) have been reported in the overwintering spruce bark beetle, Ips typographus (11).The threitol biosynthetic pathway has not been fully characterized in any organism. In humans, threitol is the major end product of xylose catabolism and is thought to be produced via the glucuronate pathway (12). Interestingly, xylans, containing predominantly β-(1→4)-linked xylose, are the major saccharide component of hemicellulose in plants (13). Thus, the wood-decomposing U. ceramboides have a potential dietary source of xylose, which could serve as the metabolic precursor of threitol.Despite the absence of experimental evidence, it has been assumed that threitol production in insects involves the PPP. In addition to being a source of 4-carbon monosaccharides, flux through the PPP generates the reducing equivalents (NADPH) needed for polyol biosynthesis (9). However, the broad specificities observed for polyol dehydrogenases (14, 15) and sugar phosphatases (16, 17) lead to the question of how high concentrations of threitol arise from the PPP without the accumulation of other C₄ and C₅ alditols. Indeed, the ability of various insect species to accumulate high concentrations of a single C₄ or C₅ alditol originating from the PPP has not been investigated (9).In this study, the threitol biosynthetic pathway in U. ceramboides has been investigated by ¹³C{¹H} NMR spectroscopy using ¹³C-labeled metabolites. The results elucidate the threitol biosynthetic pathway and lead to a new metabolic model that explains the observed selective accumulation of threitol.     

Phosphorus-31 nuclear magnetic resonance study of the thiamine diphosphate-magnesium interaction

The results obtained from a phosphorus NMR study of the interactions of Mg²⁺ with thiamine diphosphate confirm the existence of a Mg-thiamine diphosphate complex with a $\text{1}\text{1}$ stoichiometry in which the α phosphorus seems to be the most influenced by the interaction. The variations of δ and J_αβ with various concentrations of Mg²⁺ are described.     

Orthophosphate analysis by the Fiske-SubbaRow method and interference by adenosine phosphates and pyrophosphate at variable acid pH

ATP, ADP, AMP, and pyrophosphate interfere with colour development in the Fiske-SubbaRow assay procedure. The extent of interference observed is dependent on the pH of the final assay system; small changes in pH at low pH values lead to large variations in the amount of ATP interference. Addition of excess ammonium molybdate and neutralisation of any previous acid additions (such that the pH is maintained constant throughout all assay procedures) removes interference and P₁ can be estimated in the presence of relatively high ATP concentrations.     

Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida utilis: Correlation with the Conformational Stability and Activity

Invertase (β-d-fructofuranoside fructohydrolase-E.C. 3.2.1.26) is a sucrose hydrolyzing enzyme found in microbial, plant and animal sources. Invertase from Candida utilis is a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. We investigated the mechanism of stabilization of invertase with polyols (glycerol, xylitol, and sorbitol). Activity, thermodynamic and kinetic measurements of invertase were performed as a function of polyol concentration and showed that polyols act as very effective stabilizing agents. The result from the solvent-invertase interaction shows preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. Apparent thermal denaturation temperature of the protein (T _m ) rose from 75 °C to a maximum of 85 °C in 30% glycerol. The stabilization has been attributed to the preferential hydration of the enzyme.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Aerobic granulation with low strength wastewater at low aeration rate in A/O/A SBR reactor

The formation of aerobic granules with low organic loading synthetic wastewater (150-200 mg L⁻¹ of influent COD, acetate/propionate = 1/3) at low aeration rate (0.6 cm s⁻¹ of superficial gas velocity) had been investigated in the anaerobic/oxic/anoxic SBR. Aerobic granules with smooth surface and compact structure were successfully obtained after 50 days. However, these aerobic granules were unstable when the d(0.9) of granules increased to more than 1 mm. The results suggested that the aerobic granules with small diameter (smaller than 1000 μm) were more favorable for treating the low substrate loading wastewater at the low aeration rate. The cycle test revealed that most of the influent COD was removed at the anaerobic stage. The effluent concentrations of N-NH₄⁺ and P-PO₄³⁻ were lower than 1 mg L⁻¹, and the effluent concentration of nitrate gradually decreased with the granulation. Phosphate accumulating organisms were found to utilize O₂ or NO_x⁻ as electron acceptor for phosphorus removal in the study. Simultaneous nitrogen and phosphorus removal occurred inside the granules.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Acyclic polyols and higher taxa of fungi

RAST, D. M. & PFYFFER, G. E., 1989. Acyclic polyols and higher taxa of fungi. Acyclic polyols (sugar alcohols), widely distributed within the fungi, are the fungal secondary metabolites (in the sense of non-ubiquitous constituents) studied most extensively and appear to be physiologically important. An account is given of the systematic distribution of polyols within the Eumycota, and the influence of (1) the carbohydrate nutrient source, (2) the stage of development and (3) the growth form on the polyol pattern in fungi, to obtain additional insights into species constancy is considered. Delimitation of groups based on polyol characters (P, three states: P_0, polyols absent; P_1, polyols, except mannitol, present; P₂, mannitol (and other polyols) present) yields chemotaxa that coincide with Oomycetes (P_0,.), Zygo-and Hemiascomycetes (P₁), and an assemblage of taxa that consists of Chytridiomycetes, Euascomycetes, Basidiomycotina and Deuteromycotina (all P₂) with the conspicuous exception of some of the imperfect yeasts (P₁). Hence, polyol characters appear to be extremely conservative and, therefore, lend themselves to use as markers for the assignment to conventional taxa of species with doubtful systematic affinity. Considering other biochemical features, as well as the model-testing of the classification systems followed in Ainsworth & Bisby's Dictionary of the Fungi (6th and 7th editions) with the P-macrochemotaxa established here, recommendations are made to change the rank of some higher taxa, so as to render them homogeneous with respect to the P_0,., P₁ and P₂ character states. The proposals relate to Mastigomycotina, Blastomyceles and Endomycetales and are, in principle, realized already in some other classification systems.     

Polyol concentrations in Aspergillus repens grown under salt stress

Na⁺, K⁺ and the ratio of Na⁺/K⁺ were higher in cells of the halotolerant Aspergillus repens grown with 2 M NaCl than without NaCl. The osmolytes, proline, glycerol, betaine and glutamate, did not affect the Na⁺/K⁺ ratio, nor the polyol content of cells under any conditions. The concentrations of polyols, consisting of glycerol, arabitol, erythritol and mannitol, changed markedly during growth, indicating that they have a crucial role in osmotic adaptation.     

Copper toxicity to five Parmelia lichens in vitro

Treatment of Parmelia caperata, P. perlata, P. subrudecta, P. sulcata and P. tiliacea with CuSO₄ resulted in a time- and copper-concentration-dependent decrease in the total and intracellular potassium concentrations of the thallus, indicating that copper damaged the cytoplasmic membrane. Treatment with copper also resulted in a time-dependent increase in the total copper concentration of the thallus. After 4 h of exposure to copper, the process of potassium efflux was essentially completed but the absorption of copper was still increasing; moreover, the amount of copper bound to the thallus exceeded twice the amount of potassium released from the thallus, suggesting that cupric ions reached intracellular sites in the thallus, and K⁺/Cu²⁺ exchange was not electroneutral. After 5 h of exposure to copper, the extent of decrease in the total and intracellular potassium concentrations of the thallus was positively correlated with copper absorption levels, but only at 0.05<P<0.10, suggesting that membrane damage was proportional to the amount of bound copper, but other factors could have been operative, namely binding of copper to the cell wall. Acetone extracts of untreated thalli contained low concentrations of amino acids, polyols, and sugars, but considerable amounts of lichen substances: atranorin, caperatic, constictic, lecanoric, menegazziaic, protocetraric, salazinic, stictic, and usnic acids. Titration of the extracts with copper and assay of the free Cu²⁺ concentration revealed the presence of copper-binding ligands, and several successive absorption cycles, most probably corresponding to the binding of Cu²⁺ to each of the lichen substances detected in the extracts. However, no significant correlation (P>0.10) was found between the Cu²⁺-complexing capacity of acetone extracts and copper-induced membrane damage. It was concluded that in the studied Parmelia species, and in the experimental conditions used in this work, copper toxicity was not a simple function of the Cu²⁺-binding properties of the lichen substances present in the thallus. Several hypotheses were formulated to interpret the results.     

Ozone alteration of transport of cations and the Na+/K+-ATPase in human erythrocytes.

We have purified glutaminase 65-fold from cow brain; the final specific activity is 24 μmol/min/mg. The enzyme is stable between pH 7.5 and 9.0 and has maximal activity at pH 8.8. It requires P_i for activity. The dependence of activity on P_i concentration is sigmoidal; 50 mmP_i gives half-maximal velocity at pH 8.8. At 0.2 mP_i, pH 8.8, the dependence of activity on glutamine concentration is hyperbolic; the observed K_Gln was 30 mm. Increasing P_i concentrations increase the apparent V_m and decrease the apparent K_Gln. NH₄⁺ does not inhibit at concentrations up to 0.1 m. Glutamic acid inhibits competitively with respect to glutamine; at 0.2 mP_i pH 8.8, K_Gln was 30 mm and K_Glu was 19 mm. The results are consistent with a model in which NH₄⁺ is released irreversibly from the enzyme-substrate complex and is the first product released. The activity of glutaminase appears to be independent of the nature of the buffer with which it is equilibrated before being assayed.     

A New Sensitive Bioassay for Determination of Microbially Available Phosphorus in Water

The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water. However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters. Even a very low concentration of phosphorus (below 1 μg of P liter⁻¹) can promote extensive microbial growth. We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 μg of P liter⁻¹. The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of Pseudomonas fluorescens P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth. Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 ± 9,400 CFU/μg of PO₄-P. A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 μg of PO₄-P liter⁻¹. The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 μg of P liter⁻¹ (median, 0.60 μg of P liter⁻¹).