Corticosteroid-treated Rats Research Articles

A model of pulmonary toxoplasmosis in rats as potential impact on immune deficient diseases

Toxoplasma gondii is an obligate intracellular zoonotic protozoan. Pulmonary involvement is common in immunocompromised hosts but rarely occurs in immunocompetent ones. Rat pulmonary involvement could be considered as a good representative model for study of human toxoplasmosis infection to either acute toxoplasmosis, chronic toxoplasmosis, or disseminated toxoplasmosis in human immunodeficiency virus (HIV) patients. Forty Swiss-Webster rats were divided into four groups each ten rats: group 1, non-infected and untreated (normal); group 2, acute infected non-treated; and groups 3 and 4, chronic infected non-treated and corticosteroid treated respectively. Lung tissue and blood samples were taken during acute and chronic stages for parasitological and histopathological study and serological assay. Acute and chronic states revealed the presence of viable toxoplasma tachyzoites and bradyzoites in lung tissue respectively. Serological assay showed different results in acute and chronic infections. The histopathological changes in acute rat group showed that per alveolar septa has been swollen due to swelling of the pneumocystis type I and II and the lining cells of the alveoli became low cuboidal in shape. The histopathological changes in chronic untreated rat group showed that the majority of alveolar walls were thickened due to swelling of the alveolar epithelial cells and mononuclear inflammatory cells and the bronchioles due to hyperplasia of its lining epithelium. Some of the blood vessel wall contained large number of chronic Toxoplasma cysts and surrounded by mononuclear cells. While chronic corticosteroid-treated rat group showed perivascular and alveolar edema with congested per alveolar blood capillaries and alveolar edema with compensatory emphysema of some others. The study concluded that toxoplasmosis is a great of interest, serious, and often a life-threatening disease in immune-deficient patients and sometimes in immunocompetent ones. Pulmonary toxoplasmosis should be considered in the differential diagnosis in potentially HIV-infected patients with respiratory symptoms and bilateral pulmonary infiltrates. Early diagnosis and initiating specific good therapy is essential.

In vitro and in vivo activity of iclaprim, a diaminopyrimidine compound and potential therapeutic alternative against Pneumocystis pneumonia.

Pneumocystis pneumonia is a serious complication that may affect immunosuppressed patients. The absence of reliable and safe therapeutic alternatives to trimethoprim-sulfamethoxazole (TMP/SMX) justifies the search for more effective and less toxic agents. In this study, the in vitro and in vivo anti-Pneumocystis jirovecii activity of iclaprim, a diaminopyrimidine compound that exerts its antimicrobial activity through the inhibition of dihydrofolate reductase (DHFR), as does TMP, was evaluated alone or in combination with SMX. The antimicrobial activity of iclaprim was tested in vitro using an efficient axenic culture system, and in vivo using P. carinii endotracheally inoculated corticosteroid-treated rats. Animals were orally administered iclaprim (5, 25, 50mg/kg/day), iclaprim/SMX (5/25, 25/125, 50/250mg/kg/day), TMP (50mg/kg/day), or TMP/SMX (50/250mg/kg/day) once a day for ten consecutive days. The in vitro maximum effect (Emax) and the drug concentrations needed to reach 50% of Emax (EC50) were determined, and the slope of the dose-response curve was estimated by the Hill equation (Emax sigmoid model). The iclaprim EC50 value was 20.3μg/mL. This effect was enhanced when iclaprim was combined with SMX (EC50: 13.2/66μg/mL) (p = 0.002). The TMP/SMX EC50 value was 51.4/257μg/mL. In vivo, the iclaprim/SMX combination resulted in 98.1% of inhibition compared to TMP/SMX, which resulted in 86.6% of inhibition (p = 0.048). Thus, overall, the iclaprim/SMX combination was more effective than TMP/SMX both in vitro and in vivo, suggesting that it could be an alternative therapy to the TMP/SMX combination for the treatment of Pneumocystis pneumonia.

Effects of equal daily doses delivered by different power densities of low-level laser therapy at 670 nm on open skin wound healing in normal and corticosteroid-treated rats: a brief report

The optimal parameters for low-level laser therapy (LLLT) for wound healing are still open to discussion. Hence, our study was aimed at comparing the effects of different power densities of LLLT at 670 nm in rats. Four round full-thickness skin wounds were placed on the backs of 16 rats which were divided into two groups (non-steroid and steroid-treated). Three wounds were stimulated daily with a diode laser (daily dose 5 J/cm(2)) at different power densities (5, 15 and 40 mW/cm(2), respectively), and the fourth wound served as a control. Six days after surgery all animals were killed and samples removed for histological evaluation. Significant acceleration of fibroblast proliferation and new vessel formation was observed in wounds treated at the selected power densities. No significant differences were found in corticosteroid-treated rats. In conclusion, LLLT with the methodology used improved wound healing in non-steroid rats, but was not effective after corticosteroid-treatment.

Effect of equal daily doses achieved by different power densities of low-level laser therapy at 635 nm on open skin wound healing in normal and corticosteroid-treated rats

Optimal parameters of low-level laser therapy (LLLT) for wound healing are still discussed. Hence, our study was aimed to compare effects of different power densities of LLLT at 635 nm in rats. Four, round, full-thickness, skin wounds were made on the backs of 48 rats that were divided into two groups (non-steroid laser-treated and steroid laser-treated). Three wounds were stimulated daily with a diode laser (daily dose 5 J/cm(2)) each with different power density (1 mW/cm(2), 5 mW/cm(2), and 15 mW/cm(2)), whereas the fourth wound served as a control. Two days, 6 days, and 14 days after surgery, eight animals from each group were killed and samples were removed for histological evaluation. In the non-steroid laser-treated rats, significant acceleration of epithelization and collagen synthesis 2 days and 6 days after surgery was observed in stimulated wounds. In steroid laser-treated rats, 2 days and 14 days after surgery, a decreased leucocyte/macrophage ratio and a reduction in the area of granulation tissue were recorded, respectively. In conclusion, LLLT, by the method we used, improved wound healing in the non-steroid laser-treated rats, but it was useless after corticosteroid treatment.

Exploring transplacental transmission ofPneumocystisoryctolagiin first-time pregnant and multiparous rabbit does

Pneumocystis sp. is transmitted through the airborne route and presents a high host-species-specificity. Occasional reports of Pneumocystis pneumonia in still births and newborn infants suggest that other routes of transmission, e.g. transplacental might occur. The latter has been reported in rabbits but available data indicate that transplacental transmission of Pneumocystis seems not to occur in corticosteroid-treated rats and in SCID mice. The present study was undertaken to evaluate transplacental transmission of Pneumocystis oryctolagi. The spontaneously-acquired pneumocystosis rabbit model using hybrid California/New Zealand white female rabbits was selected because of similarities among rabbit and human placentas. Three different experiments were conducted in France and Chile. Pneumocystis organisms were detected by microscopy in the lungs of pregnant does and Pneumocystis DNA was found in the lungs of fetuses from the multiparous does from the second week to the end of gestation. Pneumocystis DNA was not detected in fetuses from primiparous does. Detection of Pneumocystis oryctolagi--DNA in fetuses of multiparous does and not in those of primiparous ones, suggests that transplacental transmission may be favored by multiple gestations. Whether Pneumocystis-DNA in fetal tissues from multiparous does resulted from transplacental passage of viable transmissible forms requires further investigation.

Alterations in the growth plate cartilage of rats with renal failure receiving corticosteroid therapy

Growth retardation is a complication often associated with corticosteroid therapy. Corticosteroids are frequently used in the treatment of children with chronic renal failure. To examine the effects of corticosteroids on the growth plate cartilage in renal failure, selected markers of chondrocyte function and phenotype were evaluated in the proximal tibia of subtotally nephrectomized rats treated with corticosteroid. Serum parathyroid hormone (PTH), urea nitrogen, and creatinine levels were higher in the nephrectomized animals. Weight gain was less in the corticosteroid-treated animals; however, linear growth and tibial length did not differ among the groups after 10 days of corticosteroid therapy. The total width of the growth plate and the width of the proliferative zone were much smaller in corticosteroid-treated nephrectomized (Nx-MP) animals. Type II collagen mRNA expression was lower in animals treated with corticosteroids, and proliferating-cell nuclear antigen staining, histone-4, and insulin-like growth factor-1 (IGF-1)-receptor mRNA expression were further decreased in the Nx-MP group. There was an increase in TUNEL-positive cells in the corticosteroid-treated rats with normal renal function (intact-MP), associated with an increase in Bax and a decrease in Bcl-2 protein expression. In the Nx-MP group, both Bax and Bcl-2 protein staining was much less frequent, and TUNEL-positive cells were lower in number compared with the intact-MP group. Vascular endothelial growth factor expression in the hypertrophic chondrocytes was lower in corticosteroid-treated animals. There was less gelatinase B/matrix metalloproteinase-9 expression in the Nx-MP group, which was not associated with a decrease in tartrate-resistant acid phosphatase (TRAP) staining in the chondro-osseous junction. Inhibition of chondrocyte proliferation, diminishing of apoptosis, and lower angiogenic activity may contribute to the alterations in growth plate architecture and the significant reduction in growth plate width in rats with renal failure receiving corticosteroid therapy.

Episodic corticosterone treatment accelerates kindling epileptogenesis and triggers long-term changes in hippocampal CA1 cells, in the fully kindled state.

We tested the effect of episodic (approximately 10 days) corticosterone treatment on: (i) behavioural symptoms during kindling epileptogenesis; and (ii) electrical activity in the CA1 hippocampal area during epileptogenesis, and later on, in the fully kindled state. Male rats received a corticosterone-releasing pellet (100 mg/day) shortly before kindling was started, resulting in elevated hormone levels during the early and middle stages of epileptogenesis. The appearance of moderate behavioural signs of epilepsy and severe tonic-clonic seizures was significantly accelerated in corticosterone-treated animals compared to placebo controls. During epileptogenesis, corticosterone treatment did not affect the amplitude and paired-pulse characteristics of in vivo-recorded CA1 field responses, or the duration of the afterdischarge following tetanic stimulation of the Schaffer collaterals. However, other properties of CA1 cells studied in vitro, in the fully kindled state, were altered by the earlier episodic corticosterone treatment. Thus, in kindled rats, the amplitude of the population spike in the CA1 area was significantly enhanced after prior exposure to high corticosterone levels. Prior episodic steroid treatment resulted furthermore in a significantly increased amplitude of voltage-gated Ca currents, in kindled rats. At that time, corticosterone levels of animals which had received a corticosterone-releasing pellet earlier were no longer elevated compared to the placebo controls; the corticosteroid-treated rats did also not differ from the controls with respect to the mRNA expression levels for the two corticosteroid receptor subtypes in the hippocampus. The data suggest that exposure of animals to a period of stressful experiences during a critical phase in epileptogenesis could impose lasting deleterious effects on the course of epilepsy, even when CORT levels have been normalized again.

Acute treatment with corticosteroids decreases IGF-1 and IGF-2 expression in the rat diaphragm and gastrocnemius.

Massive doses of methylprednisolone (M) or triamcinolone (T) induced diaphragmatic type IIx/b atrophy, resulting in a leftward shift of the force-frequency curve in rats (). To examine the role of insulin-like growth factors (IGFs) in these changes, IGF mRNA content was measured in costal diaphragm, gastrocnemius, and liver removed from 32 rats treated daily during 5 d either with saline (control, C and pair-fed, PF), M, or T (80 mg/kg). Blood samples were taken to measure IGF-1 serum levels. RNA levels were measured by Northern and dot-blots after hybridization with rat IGF-1 or IGF-2 cDNA probes labeled with alpha-32P. Compared with C (845 +/- 128 ng/ml), IGF-1 serum levels were significantly decreased in M (699 +/- 90 ng/ml, p < 0.001 versus C) and PF animals (505 +/- 33 ng/ml, p < 0.001 versus others) and even more so, in T-treated animals (273 +/- 134 ng/ml, p < 0.001 versus others). Along the same lines, IGF-1 expression in the liver was depressed after corticosteroid treatment and in PF, whereas IGF-2 mRNA content remained unchanged. Compared with C, the relative expression of IGF-1 mRNA in the diaphragm was depressed by 44% and 69% in the M and T groups, respectively (p < 0.0001 versus C), while it was unchanged in PF animals. In the gastrocnemius, IGF-1 expression was reduced after M and T (-51% and -59%, respectively, p < 0.0001 versus C) as well as in PF animals (-40%, p < 0.001 versus C). For IGF-2, a similar pattern of expression was found in the diaphragm and the gastrocnemius. Indeed, IGF-2 mRNA tended to decrease in corticosteroid-treated rats (NS) whereas it was unchanged in PF rats. We conclude that decreased IGF expression after corticosteroid treatment was similar in diaphragm and gastrocnemius and may be responsible for the diaphragmatic changes observed after steroid treatment.

Evaluation of drug efficacy by using animal models or in vitro systems.

The efficacy of most therapeutic and prophylactic protocols against Pneumocystis carinii pneumonia used in human patients has been tested in animal models, especially in the corticosteroid-treated rat. The advantages and drawbacks of this model have been examined in brief in Chapter 1 of this section. More recently, the nude rat, intratracheally inoculated with Pneumocystis, was used to test new anti-microbian molecules for their anti-Pneumocystis activity. In vitro systems, co-cultures of Pneumocystis with feeder cells as well as axenic cultures, were also used many times for drug screening. In this paper, the most used in vivo or in vitro drug screening systems are described. Moreover, as immunocompromised individuals, AIDS patients, especially, are often infected simultaneously by several infectious agents, a recent co-infection model is described.

XXII. Evaluation of drug efficacy by using animal models or in vitro systems

The efficacy of most therapeutic and prophylactic protocols against Pneumocystis carinii pneumonia used in human patients has been tested in animal models, especially in the corticosteroid-treated rat. The advantages and drawbacks of this model have been examined in brief in Chapter 1 of this section. More recently, the nude rat, intratracheally inoculated with Pneumocystis, was used to test new anti-microbian molecules for their anti- Pneumocystis activity. In vitro systems, co-cultures of Pneumocystis with feeder cells as well as axenic cultures, were also used many times for drug screening. In this paper, the most used in vivo or in vitro drug screening systems are described. Moreover, as immunocompromised individuals, AIDS patients, especially, are often infected simultaneously by several infectious agents, a recent co-infection model is described.

Insulin-like growth factor I increases bone formation in old or corticosteroid treated rats

We studied bone induction in subcutaneous implants of demineralized bone matrix with or without insulin-like growth factor I (IGF-I) in aged or corticosteroid-treated rats. Each rat carried one pair of implants, one control and one experiment implant, containing IGF-I dissolved in a hyaluronan solution for slow release. The rats were killed after 3 weeks and the results were evaluated by measuring the calcium content of implants. Young (6-7 weeks) and old (19-27 months) rats were used. A group of young rats was treated for 1 week with subcutaneous injections of 140 micrograms/kg dexamethasone daily. Old rats produced only approximately 1% as much bone as young rats. Local delivery of IGF-I did not increase bone formation in young rats. In old rats, bone formation was increased by IGF-I, 3000 ng/implant. Corticosteroids reduced bone formation in young rats. This effect was partially reversed by local administration of IGF-I.

Altered trimethoprim-sulfamethoxazole ratios for prophylaxis and treatment of Toxoplasma gondii and Pneumocystis carinii dual infections in rat model.

Trimethoprim-sulfamethoxazole (TMP-SMX) is the first-line drug for prophylaxis and treatment of Pneumocystis carinii pneumonia (PCP) and for prophylaxis of toxoplasmosis in HIV-infected patients. Evidence indicating intolerance related to the dose of SMX led us to examine the efficacy of altered TMP-SMX ratios in a corticosteroid-treated rat model. Infections were assessed by counting P. carinii cysts in lung and by titration of Toxoplasma gondii burdens in tissue culture. For prophylaxis, the reference regimen of 20 mg/kg TMP plus 100 mg/kg SMX was effective. Reduced doses of SMX (5 and 20 mg/kg) effective against PCP were effective against toxoplasmosis, provided the TMP dose was increased to 100 mg/kg. For curative treatment, the reversed ratio of 100 mg/kg TMP plus 20 mg/kg SMX was not effective. These results may provide a basis for altering the TMP-SMX ratios in setting of prophylaxis for both infections, especially in HIV infected patients who often poorly tolerate sulfonamides.

Pneumocystitis carinii organisms from in vitro culture are highly infectious to the nude rat.

Many in vitro systems have been used to cultivate Pneumocystis, but only limited parasite growth has been obtained by different authors. A reliable in vitro system enabling a sustained propagation of Pneumocystis appears to be an important condition for a better definition of the transmission of P. carinii pneumonia. In this work, Pneumocystis in vitro culture was performed on monolayers of L2 rat lung epithelial-like cells. Ultrastructural assessment revealed that culture parasites were structurally intact. Pneumocystis culture samples were intratracheally inoculated into corticosteroid-treated nude rats (nonlatently infected by P. carinii), which developed P. carinii pneumonia at 40 days postinoculation. The infectious power of parasites obtained in vitro was 7-10 times higher than that of parasites freshly extracted from parasitized rat lung. In summary, the present results show that it is possible to obtain in vitro highly infectious Pneumocystis forms, and this study provides a promising infectivity test for use by investigators working on Pneumocystis in vitro systems.

Use of anti-CD4+ hybridoma cells to induce Pneumocystis carinii in mice

A reduction of peripheral CD4+ cell levels has been correlated with the onset of Pneumocystis carinii pneumonia in AIDS patients. Most in vivo drug discovery and development for P. carinii have been conducted in corticosteroid-treated rats. There is need for the development of new small animal models with more selective methods of immunosuppression. This study outlines a new mouse model in which specific depletion of the CD4+ T-lymphocyte population was achieved by subcutaneous injection of G.K1.5 hybridoma cells into C3HeB/FeJ mice. A significant reduction in splenic CD4+ cells was maintained over a 10-week period following a single injection of cells. Circulating anti-CD4+ antibody was detected throughout the 10-week period in hybridoma-injected mice, while circulating antibody was undetectable 4 weeks after repeated injection of purified monoclonal antibody. There was no significant increase in the CD8+ cell populations of the hybridoma-injected mice. P. carinii cysts increased in the lungs of CD4+ T-cell-depleted mice, with the number of cysts detected comparable to levels in dexamethasone-treated mice. High levels of cysts were detected when CD4+ cell populations in the spleen remained below 5% and decreased when CD4+ populations increased above the 5% level. In mice whose CD4+ population was not reduced below 5%, there was no significant increase in P. carinii cysts detected. This study presents a new mouse model with specific immunosuppression requiring a minimum of animal manipulation for use in discovery and development of potential new therapeutics for P. carinii pneumonia.

Intratracheal interferon-gamma augments pulmonary defenses in experimental legionellosis.

To study the effects of recombinant interferon-gamma (IFN-gamma) on pulmonary defenses in vivo, we measured Ia antigen expression by alveolar macrophages and whole-lung clearance of inhaled Legionella pneumophila in normal and corticosteroid-treated rats. We found that Ia antigen was expressed by 7, 29, 50, and 65% of alveolar macrophages harvested from normal rats 24 h after intratracheal administration of 0, 10(3), 10(4), or 10(5) U of IFN-gamma, respectively, and by 76% of alveolar macrophages harvested from corticosteroid-treated rats given 10(5) U of IFN-gamma. Corticosteroid-treated rats exhibited a marked impairment in the clearance of inhaled L. pneumophila, associated with diminished release of IFN-gamma by antigen-stimulated splenocytes and blunted up-regulation of Ia antigen by alveolar-exudate macrophages. Daily intratracheal injections of IFN-gamma starting 1 day before or 1 day after infection had little effect on bacterial clearance in normal rats but markedly reduced the intrapulmonary replication of L. pneumophila and the number of bronchoalveolar neutrophils in corticosteroid-treated animals. Intraperitoneally administered IFN-gamma had no effect on Ia expression by alveolar macrophages or on bacterial clearance. IFN-gamma may be useful in the treatment of intracellular infections when targeted to the site of infection in immunosuppressed hosts.

DNA Amplification in Experimental Pneumocystosis: Characterization of Serum Pneumocystis carinii DNA and Potential P. carinii Carrier States

DNA amplification has identified P. carinii DNA in diverse biologic specimens, including the serum of patients with P. carinii pneumonia. To further examine the relationship between P. carinii DNA in serum and P. carinii infection, the corticosteroid-treated rat model of pneumocystosis was studied. By 4 weeks of immunosuppression, P. carinii DNA was detected in rat lungs and by 6 weeks, in their serum. P. carinii DNA persisted in lung tissue as long as 5 months after the withdrawal of steroids. Serum DNA disappeared 2 weeks after steroids were withdrawn. Nonimmunocompromised, sentinel rats housed near immunocompromised, P. carinii-infected rats also were studied. Within 6 weeks, P. carinii DNA became detectable in lung and by 8 weeks, in serum. P. carinii DNA disappeared rapidly from lungs and sera after sentinel rats were isolated away from corticosteroid-treated rats. These findings support the contagious transmission of P. carinii and suggest facile development of P. carinii carrier states.

Interferon-gamma Increases Alveolar Macrophage Ia Antigen Expression Despite Oral Administration of Dexamethasone to Rats

Corticosteroids have multiple effects on immune and inflammatory responses and decrease host resistance to a broad range of microorganisms. Resident tissue macrophages have been proposed as a target for the immunosuppressive effects of corticosteroids and are important in host defense against infections. During infection-induced immune responses, macrophages are activated after exposure to interferon-gamma (IFN-gamma), and class II major histocompatibility (Ia) antigens on their surface are increased. We investigated the effect of orally administered corticosteroids on alveolar macrophages, the resident macrophages of the lung parenchyma. We hypothesized that corticosteroids would inhibit the activation of alveolar macrophages and measured induction by IFN-gamma of Ia antigens as a marker of cell activation. Alveolar macrophages from normal and corticosteroid-treated rats were exposed to recombinant murine IFN-gamma (rMuIFN-gamma) in vitro and assayed for Ia transcription and surface Ia expression. Ia mRNA accumulation was induced in alveolar macrophages from normal and corticosteroid-treated rats after exposure in vitro to rMuIFN-gamma. Furthermore, rMuIFN-gamma increased surface expression of Ia proteins on alveolar macrophages from corticosteroid-treated rats, although to a lesser extent than on cells from control rats. Finally, surface Ia expression could also be increased in vivo by exposure of corticosteroid-treated rats to an aerosol containing rMuIFN-gamma. These results demonstrate that administration of oral corticosteroids, while establishing a state of immunosuppression in rats, does not abolish responsiveness of rat alveolar macrophages to rMuIFN-gamma. We speculate that IFN-gamma-induced augmentation of phagocytic cell function may constitute an important therapeutic modality to treat complications of immunodeficiency.

Impaired clearance of aerosolized Legionella pneumophila in corticosteroid-treated rats: a model of Legionnaires' disease in the compromised host.

To develop a model of legionnaires' disease in a host with defective cell-mediated immunity, rats were treated with subcutaneous cortisone acetate and exposed to aerosolized Legionella pneumophila. Bacterial clearance, histopathology, cell recovery by bronchoalveolar lavage, serology, and splenocyte blastogenesis to heat-killed L. pneumophila were studied in cortisone-treated rats and normal controls. Corticosteroid administration resulted in a dosage-related defect in the clearance of L. pneumophila. Cortisone-treated animals had a diffuse, progressive pneumonitis, but the influx of neutrophils to the lung, the serum antibody response, and the sensitization of splenocytes to L. pneumophila were not impaired by corticosteroids. The marked lymphocyte depletion observed in cortisone-treated animals may have contributed to defective expression of cell-mediated immunity. This model may be useful in further studies of the pathogenesis and treatment of legionellosis in the compromised host.

Analyses of Pneumocystis Fatty Acids

The major ester-linked fatty acids of the total lipids extracted from Pneumocystis carinii isolated from the lungs of corticosteroid-treated rats were 16:0, 18:0, 18:1, 18:2 and 20:4. Others detected included 14:0, 16:1 and 22:4. The major sphingolipid fatty acids were 16:0, 18:0, 22:0, 24:0 and 24:1; others included 14:0, 18:1, 20:0, 23:0, 24:2 and 26:0. The total lipid fatty acid compositions of preparations from appropriate lung controls were similar to those of the organism.

Postnatal changes in the distribution and elemental composition of Paneth cells in normal and corticosteroid-treated rats.

Paneth cells containing zinc-rich granules were found in the small intestine of 6-day-old rats. These cells were more numerous in older animals and were consistently most common in the distal ileum. The zinc content of granules from 10-day-old rats was similar to that found in adults (ca 300 mg atoms/kg dry weight) but no calcium could be detected. An injection of cortisone acetate at 5 days resulted in a premature increase in the numbers of Paneth cells in 10-day-old rats. The cell granules contained normal, adult levels of zinc, a calcium concentration of ca 400 mg atoms/kg dry weight and also an increased concentration of phosphorus.