... In: Homburger HA, ed. Clinical and analytical concepts in enzymology. Sko-kie, IL: College of... more ... In: Homburger HA, ed. Clinical and analytical concepts in enzymology. Sko-kie, IL: College of American Pathologists, 1982:233-56. Ektachem ... Figure 1 shows the standard curves obtained, and that we were not able to confirm the result of Yamamoto et al. ...
Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in a... more Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88-200 fold in human AIP fibroblasts and synthesis of PP was...
Hereditary coproporphyria (HCP) is an autosomal dominant disorder, resulting from a partial defic... more Hereditary coproporphyria (HCP) is an autosomal dominant disorder, resulting from a partial deficiency of the enzyme coproporphyrinogen oxidase (CPO). This enzyme catalyzes the sixth step of the heme biosynthetic pathway, and mutations in the CPO gene have been coupled to HCP. The present study was undertaken to identify disease-producing mutations in the CPOgene in nine Swedish families with HCP. Exon 1 of the CPO gene of the nine probands was analyzed directly by sequencing, and exons 2-7 were screened by denaturating gradient gel electrophoresis, followed by sequencing of exons showing abnormal band pattern. Mutations were detected in five of the nine families. In two of these families, the novel mutations 623C>T (S208F, exon 2) and 982C>T (R328C, exon 5) were identified, respectively. In the affected members of the other three families, the previously reported mutations 991C>T (R331W, exon 5) and 1339C>T (R447C, exon 7) were shown to coexist on one allele. The presen...
We determined carnitine concentrations in blood and in liver and abdominal muscle biopsy specimen... more We determined carnitine concentrations in blood and in liver and abdominal muscle biopsy specimens in 13 men and 16 women undergoing elective surgery (mostly gallbladder removal). The data suggest that the carnitine pools of plasma and erythrocytes are different. The erythrocytes show a higher acylcarnitine concentration than does plasma (P < 0.001). Several reference bases for values in tissues have been used--dry weight, noncollagen protein (NCP), and DNA--because these may be differently influenced by disease. In liver specimens, the quotient NCP (g)/DNA (g) was significantly higher in men, 54.4 +/- 6.3 (mean +/- SD), than in women, 47.7 +/- 7.0 (P < 0.01). Liver total carnitine content in relation to DNA was significantly higher in men than in women: 0.29 +/- 0.06 vs 0.22 +/- 0.08 mmol/g DNA (P < 0.01). Free carnitine content was significantly higher in men than in women independently of the reference base, e.g., 3.7 +/- 1.0 mumol/g NCP for men vs 2.9 +/- 1.0 for women ...
In experimental animals the enhancement of hepatic fatty acid oxidation and ketogenic capacity is... more In experimental animals the enhancement of hepatic fatty acid oxidation and ketogenic capacity is accompanied by a rise in the concentration of liver carnitine. Massive obesity is characterized by enhanced fatty acid turnover, insulin resistance, and often a fatty liver. Carnitine concentrations were determined in liver, abdominal muscle tissue, and blood in morbidly obese women. The liver and muscle carnitine concentrations were significantly higher in the obese subjects than in the lean control subjects. These findings suggest an increase of the whole-body carnitine pool. In the obese subjects there was also a significant positive correlation between liver and muscle carnitine concentrations. In the majority of the obese subjects fatty changes could be demonstrated in the liver. The plasma insulin concentration tended to be positively correlated with the degree of fat infiltration and negatively correlated with the liver carnitine content. It is concluded that the liver carnitine ...
A spectrophotometric method for carnitine has been adapted to the Cobas Bio centrifugal analyzer.... more A spectrophotometric method for carnitine has been adapted to the Cobas Bio centrifugal analyzer. The addition of carnitine to a system containing carnitine acetyltransferase (EC 2.3.1.7) and acetyl-CoA gives rise to the formation of CoA. The system is coupled to 5,5'-dithiobis(2-nitrobenzoate) (DTNB). Assay response varied linearly with concentration of carnitine over a wide concentration range. The total CV was 5.5% for a carnitine concentration in serum of 58.0 mumol/L. Analytical recovery of carnitine added to a serum sample was 93%. No interference was found in icteric, not grossly hemolyzed, lipemic, or uremic sera. Comparison with a radioenzymatic method showed that results correlated well (r greater than 0.965) but the present method gave values proportionally greater by 10 to 25% for samples of plasma, dialysis fluid, urine, and muscle tissue. Advantages over the original spectrophotometric assays involving DTNB include low reagent costs, rapidity, simplicity, and repro...
Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinoge... more Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinogen deaminase (PBGD), the 3rd enzyme in heme synthesis. It is clinically characterized by acute attacks of neuropsychiatric symptoms and biochemically by increased urinary excretion of the porphyrin precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA). A mouse model that is partially deficient in PBGD and biochemically mimics AIP after induction of the hepatic ALA synthase by phenobarbital was used in this study to identify the site of formation of the presumably toxic porphyrin precursors and study the effect of enzyme-replacement therapy by using recombinant human PBGD (rhPBGD). After 4 d of phenobarbital administration, high levels of PBG and ALA were found in liver, kidney, plasma, and urine of the PBGD-deficient mice. The administration of rhPBGD intravenously or subcutaneously after a 4-d phenobarbital induction was shown to lower the PBG level in plasma in a dose-depende...
Barbaro M, Kotajärvi M, Harper P, Floderus Y. Identification of an AluY‐mediated deletion of exon... more Barbaro M, Kotajärvi M, Harper P, Floderus Y. Identification of an AluY‐mediated deletion of exon 5 in the CPOX gene by MLPA analysis in patients with hereditary coproporphyria.Hereditary coproporphyria (HCP) is an autosomal dominantly inherited hepatic porphyria, caused by a mutation in the coproporphyrinogen oxidase (CPOX) gene. The genetic defect leads to a partial defect of CPOX, the sixth enzyme involved in haem biosynthesis. Affected individuals can develop acute life‐threatening attacks of neurovisceral symptoms and/or more rarely cutaneous symptoms such as skin fragility and blistering. The identification of the genetic defect in HCP families is of crucial importance to detect the carrier status which allows counselling to prevent possible triggering factors, e.g. certain drugs, alcohol, or fasting. In a total of nine Swedish HCP families, routine gene sequence analysis had identified a causative mutation in only five. In the present study, using an in‐house developed synthe...
Scandinavian Journal of Clinical & Laboratory Investigation, 2002
Acute intermittent porphyria (AIP) is an inborn error of heme synthesis in which the third enzyme... more Acute intermittent porphyria (AIP) is an inborn error of heme synthesis in which the third enzyme, porphobilinogen deaminase (PBGD), is deficient. The disease is characterized by recurrent attacks of acute abdominal pain often accompanied by neuropsychiatric symptoms. Current therapeutic treatment with heme is only palliative and no curative alternative exists. The present report describes the first step towards a gene therapy treatment for AIP. Mouse cDNA encoding the PBGD enzyme was cloned and four vectors containing the full-length mouse and human cDNA of the housekeeping and erythroid PBGD isoforms under the control of a cytomegalovirus promoter were constructed. The vectors, condensed to polyethylenimine, were successfully transfected to NIH 3T3 and HeLa cells as determined by enzymatic activity measurements. Thus, the PBGD activity was increased 3-10 times in NIH 3T3 cells and 95-240 times in HeLa cells. The expression was shown to be dose and time dependent, with the highest level of activity observed in HeLa cells after 72 h posttransfection. Non-viral gene transfer was also undertaken in PBGD-deficient fibroblasts established from an AIP patient. Complete normalization of the PBGD activity was accomplished after the addition of 2.5 microg DNA per well. Further addition of DNA increased the PBGD activity up to threefold the normal value. The study documents a successful gene transfer and a high degree of PBGD expression in different cell-lines, indicating a potential for future gene therapy in AIP.
Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient act... more Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyria include episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenous heme administration is palliative and there is no way to prevent the attacks. Thus, efforts are focused on methods to replace the deficient activity in the liver to prevent the acute attacks of this hepatic porphyria. Here we explore the efficiency of a non-viral gene delivery to obtain PBGD expression in the liver of AIP transgenic mice. Four vectors were evaluated: naked DNA and DNA complexed to liposomes, polyethylenimine (PEI), and PEI-galactose, using a luciferase construct as reporter gene. The vectors were administered intravenously or directly into the portal vein with transient blood flow blockage. After tail vein injection of the DNA complexes, the liposome vector had the highest luciferase expression in lung and less in liver. When injected into the portal vein, the naked DNA had considerably higher hepatic reporter gene expression; 100 microg of naked DNA had the highest hepatic luciferase expression 24h after portal vein injection. When these vectors were used to deliver the PBGD gene into the AIP mouse model no enhancement of the endogenous PBGD activity in liver was detectable, despite the presence of the PBGD-plasmids as verified by PCR. Thus, more efficient non-viral vectors are needed to express sufficient PBGD activity over the endogenous hepatic level (approximately 30% of normal) in this murine system.
... In: Homburger HA, ed. Clinical and analytical concepts in enzymology. Sko-kie, IL: College of... more ... In: Homburger HA, ed. Clinical and analytical concepts in enzymology. Sko-kie, IL: College of American Pathologists, 1982:233-56. Ektachem ... Figure 1 shows the standard curves obtained, and that we were not able to confirm the result of Yamamoto et al. ...
Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in a... more Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88-200 fold in human AIP fibroblasts and synthesis of PP was...
Hereditary coproporphyria (HCP) is an autosomal dominant disorder, resulting from a partial defic... more Hereditary coproporphyria (HCP) is an autosomal dominant disorder, resulting from a partial deficiency of the enzyme coproporphyrinogen oxidase (CPO). This enzyme catalyzes the sixth step of the heme biosynthetic pathway, and mutations in the CPO gene have been coupled to HCP. The present study was undertaken to identify disease-producing mutations in the CPOgene in nine Swedish families with HCP. Exon 1 of the CPO gene of the nine probands was analyzed directly by sequencing, and exons 2-7 were screened by denaturating gradient gel electrophoresis, followed by sequencing of exons showing abnormal band pattern. Mutations were detected in five of the nine families. In two of these families, the novel mutations 623C>T (S208F, exon 2) and 982C>T (R328C, exon 5) were identified, respectively. In the affected members of the other three families, the previously reported mutations 991C>T (R331W, exon 5) and 1339C>T (R447C, exon 7) were shown to coexist on one allele. The presen...
We determined carnitine concentrations in blood and in liver and abdominal muscle biopsy specimen... more We determined carnitine concentrations in blood and in liver and abdominal muscle biopsy specimens in 13 men and 16 women undergoing elective surgery (mostly gallbladder removal). The data suggest that the carnitine pools of plasma and erythrocytes are different. The erythrocytes show a higher acylcarnitine concentration than does plasma (P < 0.001). Several reference bases for values in tissues have been used--dry weight, noncollagen protein (NCP), and DNA--because these may be differently influenced by disease. In liver specimens, the quotient NCP (g)/DNA (g) was significantly higher in men, 54.4 +/- 6.3 (mean +/- SD), than in women, 47.7 +/- 7.0 (P < 0.01). Liver total carnitine content in relation to DNA was significantly higher in men than in women: 0.29 +/- 0.06 vs 0.22 +/- 0.08 mmol/g DNA (P < 0.01). Free carnitine content was significantly higher in men than in women independently of the reference base, e.g., 3.7 +/- 1.0 mumol/g NCP for men vs 2.9 +/- 1.0 for women ...
In experimental animals the enhancement of hepatic fatty acid oxidation and ketogenic capacity is... more In experimental animals the enhancement of hepatic fatty acid oxidation and ketogenic capacity is accompanied by a rise in the concentration of liver carnitine. Massive obesity is characterized by enhanced fatty acid turnover, insulin resistance, and often a fatty liver. Carnitine concentrations were determined in liver, abdominal muscle tissue, and blood in morbidly obese women. The liver and muscle carnitine concentrations were significantly higher in the obese subjects than in the lean control subjects. These findings suggest an increase of the whole-body carnitine pool. In the obese subjects there was also a significant positive correlation between liver and muscle carnitine concentrations. In the majority of the obese subjects fatty changes could be demonstrated in the liver. The plasma insulin concentration tended to be positively correlated with the degree of fat infiltration and negatively correlated with the liver carnitine content. It is concluded that the liver carnitine ...
A spectrophotometric method for carnitine has been adapted to the Cobas Bio centrifugal analyzer.... more A spectrophotometric method for carnitine has been adapted to the Cobas Bio centrifugal analyzer. The addition of carnitine to a system containing carnitine acetyltransferase (EC 2.3.1.7) and acetyl-CoA gives rise to the formation of CoA. The system is coupled to 5,5'-dithiobis(2-nitrobenzoate) (DTNB). Assay response varied linearly with concentration of carnitine over a wide concentration range. The total CV was 5.5% for a carnitine concentration in serum of 58.0 mumol/L. Analytical recovery of carnitine added to a serum sample was 93%. No interference was found in icteric, not grossly hemolyzed, lipemic, or uremic sera. Comparison with a radioenzymatic method showed that results correlated well (r greater than 0.965) but the present method gave values proportionally greater by 10 to 25% for samples of plasma, dialysis fluid, urine, and muscle tissue. Advantages over the original spectrophotometric assays involving DTNB include low reagent costs, rapidity, simplicity, and repro...
Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinoge... more Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinogen deaminase (PBGD), the 3rd enzyme in heme synthesis. It is clinically characterized by acute attacks of neuropsychiatric symptoms and biochemically by increased urinary excretion of the porphyrin precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA). A mouse model that is partially deficient in PBGD and biochemically mimics AIP after induction of the hepatic ALA synthase by phenobarbital was used in this study to identify the site of formation of the presumably toxic porphyrin precursors and study the effect of enzyme-replacement therapy by using recombinant human PBGD (rhPBGD). After 4 d of phenobarbital administration, high levels of PBG and ALA were found in liver, kidney, plasma, and urine of the PBGD-deficient mice. The administration of rhPBGD intravenously or subcutaneously after a 4-d phenobarbital induction was shown to lower the PBG level in plasma in a dose-depende...
Barbaro M, Kotajärvi M, Harper P, Floderus Y. Identification of an AluY‐mediated deletion of exon... more Barbaro M, Kotajärvi M, Harper P, Floderus Y. Identification of an AluY‐mediated deletion of exon 5 in the CPOX gene by MLPA analysis in patients with hereditary coproporphyria.Hereditary coproporphyria (HCP) is an autosomal dominantly inherited hepatic porphyria, caused by a mutation in the coproporphyrinogen oxidase (CPOX) gene. The genetic defect leads to a partial defect of CPOX, the sixth enzyme involved in haem biosynthesis. Affected individuals can develop acute life‐threatening attacks of neurovisceral symptoms and/or more rarely cutaneous symptoms such as skin fragility and blistering. The identification of the genetic defect in HCP families is of crucial importance to detect the carrier status which allows counselling to prevent possible triggering factors, e.g. certain drugs, alcohol, or fasting. In a total of nine Swedish HCP families, routine gene sequence analysis had identified a causative mutation in only five. In the present study, using an in‐house developed synthe...
Scandinavian Journal of Clinical & Laboratory Investigation, 2002
Acute intermittent porphyria (AIP) is an inborn error of heme synthesis in which the third enzyme... more Acute intermittent porphyria (AIP) is an inborn error of heme synthesis in which the third enzyme, porphobilinogen deaminase (PBGD), is deficient. The disease is characterized by recurrent attacks of acute abdominal pain often accompanied by neuropsychiatric symptoms. Current therapeutic treatment with heme is only palliative and no curative alternative exists. The present report describes the first step towards a gene therapy treatment for AIP. Mouse cDNA encoding the PBGD enzyme was cloned and four vectors containing the full-length mouse and human cDNA of the housekeeping and erythroid PBGD isoforms under the control of a cytomegalovirus promoter were constructed. The vectors, condensed to polyethylenimine, were successfully transfected to NIH 3T3 and HeLa cells as determined by enzymatic activity measurements. Thus, the PBGD activity was increased 3-10 times in NIH 3T3 cells and 95-240 times in HeLa cells. The expression was shown to be dose and time dependent, with the highest level of activity observed in HeLa cells after 72 h posttransfection. Non-viral gene transfer was also undertaken in PBGD-deficient fibroblasts established from an AIP patient. Complete normalization of the PBGD activity was accomplished after the addition of 2.5 microg DNA per well. Further addition of DNA increased the PBGD activity up to threefold the normal value. The study documents a successful gene transfer and a high degree of PBGD expression in different cell-lines, indicating a potential for future gene therapy in AIP.
Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient act... more Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyria include episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenous heme administration is palliative and there is no way to prevent the attacks. Thus, efforts are focused on methods to replace the deficient activity in the liver to prevent the acute attacks of this hepatic porphyria. Here we explore the efficiency of a non-viral gene delivery to obtain PBGD expression in the liver of AIP transgenic mice. Four vectors were evaluated: naked DNA and DNA complexed to liposomes, polyethylenimine (PEI), and PEI-galactose, using a luciferase construct as reporter gene. The vectors were administered intravenously or directly into the portal vein with transient blood flow blockage. After tail vein injection of the DNA complexes, the liposome vector had the highest luciferase expression in lung and less in liver. When injected into the portal vein, the naked DNA had considerably higher hepatic reporter gene expression; 100 microg of naked DNA had the highest hepatic luciferase expression 24h after portal vein injection. When these vectors were used to deliver the PBGD gene into the AIP mouse model no enhancement of the endogenous PBGD activity in liver was detectable, despite the presence of the PBGD-plasmids as verified by PCR. Thus, more efficient non-viral vectors are needed to express sufficient PBGD activity over the endogenous hepatic level (approximately 30% of normal) in this murine system.
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Papers by Pauline Harper