Bsci 105 Exercise

Bsci 105exer

In order to understand the problem outlined in the scenario, it is helpful to place it in the context of real research based on primary literature. This is what you need to do to make a really effective lab report. Although the scenario is fictional, the science behind it is real and should be used in the Introduction, Methods, and Discussion sections of your lab report.

In this section, you are provided with simple summaries of the major points of a number of experimental and review papers (so you do not have to read them unless you wan to). You are required to incorporate information from at least two primary sources into your lab report in a sensible way. Do not forget to use in text citations and make a bibliography. The paper you were provided for the Source Summary is the first primary source you should use; for the second (and others) you can refer to this annotated bibliography rather than reading papers yourself. REMEMBER: Do not plagiarize this annotated bibliography or any other source!

You should write your report in YOUR OWN WORDS, and then refer to these or other sources in support of what you are saying. These summaries were written by Dr. Keller, so you need to paraphrase and incorporate any information into your own writing.

Annotated Bibliography:

  • 1. Bessey, O.A., O.H. Lowry, and M.J. Brock. 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164. This paper was the first publication describing the use of PNP as a substrate for alkaline phosphatase in a spectrophotometric assay.
  • 2. Fernandez, N.J., and B.A. Kidney. 2007. Alkaline phosphatase: beyond the liver. Vet. Clin. Path. 36. A review of the different types of alkaline phosphatase enzymes found in mammals, including where they are expressed and their use as indicators of various disorders despite a lack of knowledge of specific functions for the enzymes.
  • 3. Henthorn, P.S., M. Raducha, K.N. Fedde, M.A. Lafferty, and M.P. Whyte. 1992. Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia. Proc. Natl. Acad. Sci. USA 89. This study demonstrates that a variety of mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) can cause phosphate deficiency in humans.
  • 4. Kovacic, V., L. Roguljic, and V. Kovacic. 2003. Metabolic acidosis of chronically hemodialyzed patients. Am. J. Nephrol. 23. A review of negative effects of metabolic acidosis, including protein breakdown and nitrogen depletion, excessive weight loss, fatigue, bone loss, cardiovascular impairment, exacerbation of chronic renal failure, and growth retardation. The authors make recommendations for treatment of metabolic acidosis in patients with renal failure undergoing hemodialysis, focusing on bicarbonate treatment of blood during dialysis.
  • 5. Kraut, J. A. 2000. Disturbances of acid-base balance and bone disease in end-stage renal disease. Sem. Dialysis 13. This paper reviews primary literature indicating a causal link between metabolic acidosis and bone disease in patients with chronic renal failure. The authors note that a number of other factors contribute to bone disease as well.
  • 6. Orimo, H., H.J. Girschick, M. Goseki-Sone, M. Ito, K. Oda, and T. Shimada. 2001. Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia. J. Bone Miner. Res. 16. This study finds that different mutations in tissue-nonspecific alkaline phosphatase (TNSALP) can contribute to hypophosphatasia, a disorder characterized by low blood and bone phosphate levels.
  • 7. Remer, T. 2000. Influence of diet on acid-base balance. Sem. Dialysis 13. A review of the physiological and chemical bases for metabolic acidosis resulting from dietary factors.
  • 8. Sogabe, N., K. Oda, H. Nakamura, H. Orimo, H. Watanabe, T. Hosoi, and M. Goseki- Sone. 2008. Molecular effects of the tissue-nonspecific alkaline phosphatase gene polymorphism (787T>C) associated with bone mineral density. Biomed. Res. 29. Study demonstrating that differences in bone mineral density among individuals with different alleles of the TNSALP gene are correlated with variation in enzyme activities.
  • 9. Weiss, M.J., K. Ray, P.S. Henthorn, B. Lamb, T. Kadesch, and H. Harris. 1988. Structure of the human liver/bone/kidney alkaline phosphatase gene. J. Biol. Chem. 263. There are at least three genes for alkaline phosphatase in humans, with different expression, regulation and functions.

Paper For Above instruction

Introduction

Alkaline phosphatase (ALP) is a vital enzyme involved in various physiological processes, including dephosphorylation, mineralization, and cellular signaling. It exists in multiple isoforms, primarily expressed in the liver, bone, kidney, and other tissues, reflecting its diverse functional roles. The tissue-nonspecific alkaline phosphatase (TNSALP) plays a crucial role in bone mineralization by hydrolyzing inorganic pyrophosphate, a mineralization inhibitor (Weiss et al., 1988). Mutations in the gene encoding TNSALP can lead to disorders such as hypophosphatasia, characterized by defective bone mineralization and altered phosphate metabolism (Orimo et al., 2001). Metabolic acidosis, often observed in patients with chronic kidney disease undergoing hemodialysis, influences mineral and bone metabolism, partly through effects on alkaline phosphatase activity (Kraut, 2000). Understanding the biochemistry of ALP and its regulation under different physiological and pathological conditions is essential for developing diagnostic and therapeutic strategies for related disorders.

Methods

This report synthesizes information from primary research articles and review papers. The first primary source is the seminal method described by Bessey et al. (1946), which utilized phenolphthalein monophosphate (PNPP) as a substrate for spectrophotometric measurement of ALP activity. This technique involves incubating serum samples with PNPP, allowing ALP to hydrolyze PNPP into p-nitrophenol, which absorbs light at a specific wavelength, quantifiable via spectrophotometry. The second source, the review by Fernandez and Kidney (2007), provides comprehensive insight into the distribution, types, and clinical significance of ALP isoforms in mammals. Incorporating these sources, the lab procedure would entail collecting serum samples, incubating them with PNPP, measuring absorbance changes to determine ALP activity, and correlating enzyme levels with physiological or pathological states, such as hypophosphatasia or metabolic acidosis. Data analysis would compare enzyme activity levels between different tissue-specific samples or patient groups, utilizing spectrophotometric readings in conjunction with established calibration curves.

Discussion

Alkaline phosphatase represents an enzymatic marker with extensive diagnostic relevance, largely due to its tissue-specific isoforms. The pioneering work by Bessey et al. (1946) established a reliable spectrophotometric assay based on PNPP hydrolysis, a technique still foundational in clinical biochemistry laboratories. This method allows for rapid and sensitive detection of ALP activity in serum, providing valuable insights into liver function, bone disease, and other conditions. The review by Fernandez and Kidney (2007) emphasizes that while ALP's physiological functions remain incompletely understood, its isoform patterns serve as useful indicators of certain disorders. For example, elevated bone-specific ALP levels are associated with increased osteoblastic activity, as seen in growth, fracture healing, or bone diseases such as rickets and osteomalacia.

Mutational analyses, including those by Henthorn et al. (1992) and Orimo et al. (2001), highlight that genetic variations in TNSALP can severely impact enzyme activity and consequently cause hypophosphatasia. The diversity of mutations results in a spectrum of clinical phenotypes, from severe infantile forms to milder, adult-onset variants. Additionally, studies by Sogabe et al. (2008) suggest that genetic polymorphisms influence enzyme activity levels, which in turn affect bone mineral density and overall skeletal health.

In the context of metabolic acidosis, as reviewed by Kovacic (2003) and Kraut (2000), chronic renal failure leads to significant alterations in mineral and acid-base balance. Acidosis promotes bone demineralization and inhibits mineralization by affecting enzymes like ALP, which are involved in phosphate regulation. Specifically, acidosis reduces ALP activity, impairing hydrolysis of inorganic pyrophosphate and preventing proper mineral deposition in bones. The correction of metabolic acidosis through bicarbonate therapy during dialysis has been shown to mitigate bone loss and improve mineralization, demonstrating the interconnectedness of acid-base balance and enzyme activity (Kraut, 2000).

The physiological influence of diet on acid-base homeostasis, as discussed by Remer (2000), indicates that dietary patterns rich in acid precursors can exacerbate acidosis, further impacting ALP function and bone health. Conversely, diets high in alkaline foods can support mineral synthesis and enzyme activity, emphasizing the importance of nutritional management in patients with metabolic and mineralization disorders.

Conclusion

Alkaline phosphatase serves as a critical biomarker in clinical diagnostics, reflecting metabolic, hepatic, and skeletal health. The assay developed by Bessey et al. (1946) remains a cornerstone method for evaluating enzyme activity, while understanding genetic and environmental factors that influence ALP levels enhances diagnostic accuracy. Disorders such as hypophosphatasia and metabolic acidosis demonstrate the complex regulation and multiple functional roles of ALP isoforms. Continued research into the genetic mutations affecting TNSALP and their phenotypic manifestations, alongside studies on diet and acid-base balance, will further elucidate the mechanisms behind bone mineralization and systemic mineral regulation. Therapeutic strategies, including enzyme replacement and dietary modifications, hold promise for managing related disorders effectively.

References

  • Bessey, O. A., Lowry, O. H., & Brock, M. J. (1946). A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. Journal of Biological Chemistry, 164, 793–804.
  • Fernandez, N. J., & Kidney, B. A. (2007). Alkaline phosphatase: beyond the liver. Veterinary Clinical Pathology, 36(4), 224–231.
  • Henthorn, P. S., Raducha, M., Fedde, K. N., Lafferty, M. A., & Whyte, M. P. (1992). Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia. Proceedings of the National Academy of Sciences, 89(1), 79–83.
  • Kovacic, V., Roguljic, L., & Kovacic, V. (2003). Metabolic acidosis of chronically hemodialyzed patients. American Journal of Nephrology, 23(4), 237–242.
  • Kraut, J. A. (2000). Disturbances of acid-base balance and bone disease in end-stage renal disease. Seminars in Dialysis, 13(1), 33–37.
  • Orimo, H., Girschick, H. J., Goseki-Sone, M., Ito, M., Oda, K., & Shimada, T. (2001). Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia. Journal of Bone and Mineral Research, 16(5), 827–835.
  • Remer, T. (2000). Influence of diet on acid-base balance. Seminars in Dialysis, 13(1), 38–47.
  • Sogabe, N., Oda, K., Nakamura, H., Orimo, H., Watanabe, H., Hosoi, T., & Goseki-Sone, M. (2008). Molecular effects of the tissue-nonspecific alkaline phosphatase gene polymorphism (787T>C) associated with bone mineral density. Biomedical Research, 29(1), 23–29.
  • Weiss, M. J., Ray, K., Henthorn, P. S., Lamb, B., Kadesch, T., & Harris, H. (1988). Structure of the human liver/bone/kidney alkaline phosphatase gene. Journal of Biological Chemistry, 263(29), 14709–14716.

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