Jon A. Van Loon

Pharmacogenetics of acute azathioprine toxicity: Relationship to thiopurine methyltransferase genetic polymorphism

Azathioprine therapy can cause acute myelosuppression. Toxicity is in part caused by the incorporation of azathioprine‐derived 6‐thioguanine nucleotides (6‐TGN) into deoxyribonucleic acid (DNA). The enzyme thiopurine methyltransferase (TPMT) plays an important role in azathioprine catabolism. TPMT activity is controlled by a common genetic polymorphism, and one in 300 subjects has very low enzyme activity. Azathioprine was withdrawn in five study patients because of acute myelosuppression. The duration of azathioprine treatment was 21 to 70 days (median, 28), and the daily oral dose was 1.0 to 2.5 mg/kg. Sixteen control patients who had been taking oral azathioprine (1.1 to 2.0 mg/kg daily for more than 6 months) with no history of myelosuppression were studied. All subjects had normal liver and kidney function. When compared with the control group, the five patients with myelosuppression had very low TPMT activities and abnormally high 6‐TGN concentrations. Inherited low TPMT activity appears to be a major risk factor for acute azathioprine‐induced myelosuppression.

Thermolabile and thermostable human platelet phenol sulfotransferase - Substrate specificity and physical separation

SummaryHuman platelets contain at least two forms of phenol sulfotransferase (PST), a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which micromolar concentrations of phenol can serve as substrate. At higher concentrations phenol is also a substrate for the TL form. Studies of the regulation and the possible clinical value of measurements of platelet PST have been hampered because there is no specific substrate for the TS form of the enzyme. The purposes of these experiments were to determine whether there might be a better substrate than phenol for use in measurement of the activity of the TS form of platelet PST, and to attempt to physically separate the two forms of the platelet enzyme. The results of substrate kinetic, thermal stability, and inhibitor studies performed with platelet homogenates were all compatible with the conclusion that p-nitrophenol and 6-OH-melatonin were substrates for both the TS and TL forms of platelet PST. Norepinephrine, epinephrine and 5-OH-tryptamine were substrates for only the TL form. The apparent Km constants of the two forms of PST for p-nitrophenol differed by 7,100-fold when measured in platelet homogenates. This difference was 200 times greater than that which has been reported for phenol. Therefore, p-nitrophenol is the preferred substrate for measurement of the TS PST activity if interference by the TL activity is to be avoided. This information made it possible to use p-nitrophenol as a substrate in experiments designed to separate the two forms of platelet PST. The TS and TL forms of the platelet enzyme were separated by ion exchange chromatography. Experiments performed with the partially purified and separated forms confirmed that p-nitrophenol, phenol, 6-OH-melatonin and acetaminophen were substrates for both forms. Apparent Km constants of the two forms differed most for p-nitrophenol.

Thiopurine methyltransferase biochemical genetics: Human lymphocyte activity

The level of human erythrocyte (RBC) thiopurine methyltransferase (TPMT) activity is inherited as a monogenic trait. Experiments were performed to determine whether the level of TPMT activity in the human lymphocyte is regulated in parallel with RBC TPMT. Supernatants of lymphocyte homogenates contained TPMT activity. Lymphocyte TPMT activity was maximal at a reaction pH of 6.6. The apparent Km value for 6-mercaptopurine, the thiopurine substrate for the reaction, was 8.1×10−4m, and the apparent Km value for S-adenosyl-l-methionine, the methyl donor for the reaction, was 3.6×10−6m. The average TPMT activity in lymphocytes isolated from blood of 55 randomly selected subjects was 11.0±0.4 units/109 cells (mean ± SE), with a range of from 4.8 to 17.7 units/109 cells. There was a significant correlation of relative RBC with relative lymphocyte TPMT activity in blood samples from these 55 subjects, with a correlation coefficient of 0.563 (P<0.001). The correlation coefficient for RBC with platelet enyzme activities in these same subjects was also highly significant (r=0.680, P<0.001). Blood samples from four previously identified subjects who were homozygous for the allele TPMTL, subjects who lacked detectable RBC enzyme activity, also lacked detectable lymphocyte and platelet TPMT activities. These results were compatible with the conclusion that the genetic polymorphism which regulates RBC TPMT activity also regulates the level of human lymphocyte and platelet TPMT activities.

Purine substrates for human thiopurine methyltransferase

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). A genetic polymorphism regulating TPMT activity in human tissue is an important factor responsible for individual differences in the toxicity and therapeutic efficacy of these drugs. Because of the clinical importance of this polymorphism, we studied 18 purine derivatives, including ribonucleosides and ribonucleotides, as potential substrates for purified human kidney TPMT. Sixteen of the compounds studied were substrates for the enzyme, with Km values that varied from 29.1 to 1270 microM and with Vmax values that varied from 75 to 2340 U/mg protein. The thiopurines tested had Km values that were uniformly lower than were those of the corresponding ribonucleosides or ribonucleotides. 6-Selenopurine derivatives had the lowest Km values of the compounds studied. Finally, oxidized purines with an OH in the 8-position were methylated by the enzyme, but 2-OH compounds were potent inhibitors of TPMT.

Human platelet phenol sulfotransferase: Familial variation in thermal stability of the TS form

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catechol and phenolic drugs and xenobiotic compounds. Platelets and other tissues contain at least two forms of PST, forms that have been designated the “TL” and the “TS” forms. We measured the thermal stability of platelet TS PST in blood samples from 218 randomly selected unrelated subjects by heating platelet homogenates at 44° C for 15 min. Thermal stability was expressed as the ratio of the enzyme activity remaining after preincubation to that in an unheated sample, a heated/control (H/C) ratio. The frequency distribution of H/C ratios for this population sample was bimodal, with a nadir at an H/C ratio of 0.33. Of the 218 subjects studied, 29 (13.3%) had thermolabile TS PST (H/C<0.33). Platelet samples were then obtained from subjects with thermolabile and thermostable TS platelet PST. PST activity in these platelet samples had similar apparent Km constants for substrates. IC50 values for inhibition of TS PST by 2,6-dichloro-4-nitrophenol in these samples were also nearly identical. The results of experiments in which platelet homogenates from subjects with thermolabile and thermostable TS PST were mixed and the results of experiments in which platelet homogenates were subjected to gel filtration chromatography were compatible with the conclusion that individual differences in TS PST thermal stability were properties of PST itself. Finally, there was a significant familial aggregation of the trait of thermolabile TS PST when H/C ratios were measured in platelet homogenates from 231 members of 49 randomly selected families.

Pharmacogenetics of N‐methylation: Heritability of human erythrocyte histamine N‐methyltransferase activity

Histamine N‐methyltransferase (HNMT) catalyzes the Nτ‐methylation of histamine. HNMT is present in many human tissues, including the red blood cell (RBC). Our study evaluated the possible role of inheritance in the regulation of individual variations in human RBC HNMT activity. HNMT activity was measured in RBC lysates from 241 members of 51 nuclear families. After correction for the gender‐specific effects of age, the frequency distribution of RBC HNMT activities was unimodal, and activities varied threefold within 2 SDs of the mean. The correlation of HNMT activities in RBCs from 45 pairs of spouses was only 0.070, indicating that shared environment did not result in similar activities among genetically unrelated individuals. Correlation coefficients were also calculated for pairs of genetically related individuals. All of these correlations were significant except the mother–oldest son correlation. The majority of the correlations did not differ significantly from those predicted for a trait with a heritability of 1.0 (100%). Our results demonstrate a significant familial aggregation of human RBC HNMT activity and suggest that inheritance may play an important role in the regulation of variation in the activity of this N‐methyltransferase enzyme in the human RBC.

Thiol methylation pharmacogenetics: Heritability of human erythrocyte thiol methyltransferase activity

Thiol methylation of aliphatic sulfhydryl drugs is catalyzed by thiol methyltransferase (TMT), an enzyme activity that can be measured in the human erythrocyte (RBC) membrane. As a first step toward determining the possible role of inheritance in the regulation of individual variations in the S‐methylation of drugs in man, the heritability of human RBC membrane TMT activity was determined. RBC TMT activity was measured in blood samples from 231 first‐degree relatives in 47 randomly selected families. The frequency distribution of enzyme activities was unimodal, with a fivefold variation within ±2 SDs. RBC TMT activity did not correlate with either age or sex. Heritability in the “narrow” sense (h2) was estimated by comparing correlations of RBC TMT activities in first‐degree relatives with theoretical values expected for a trait under total additive genetic control. The correlation between RBC TMT activities in mothers and fathers in these families was only 0.04, a finding that made shared environment a less likely explanation for significant correlations among other family members. However, sibling‐sibling (S‐S), parent‐offspring (P‐O), and midparent (average of two parental values)‐offspring (M‐O) correlations were 0.49, 0.49, and 0.69. Theoretical expected values for a trait with a heritability of 1.0 (100%), for these same correlations would be 0.5, 0.5, and 0.71. Estimates of h2 calculated from the observed correlations were 0.98 (S‐S), 0.98 (P‐O), and 0.98 (M‐O). None of the calculated values for heritability differed significantly from the theoretical maximum value of 1.0. These results suggested that inheritance was the primary factor regulating the fivefold variation in RBC TMT activity.

Sulfation pharmacogenetics: correlation of human platelet and small intestinal phenol sulfotransferase

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic drugs. All human tissues studied contain a thermostable (TS) form of PST, which catalyzes the sulfate conjugation of “simple” phenols such as p‐nitrophenol, and a thermolabile (TL) form, which catalyzes the sulfation of dopamine and other monoamines. In the present study we tested the hypothesis that genetically controlled levels of TS and TL PST activity in the platelet, as well as inherited variations in the thermal stability of platelet TS PST, might reflect those same characteristics of the enzyme in a less accessible tissue, human small intestinal mucosa. Platelet TS and TL PST activities and TS PST thermal stability were measured in blood samples from 45 randomly selected healthy subjects, and 14 of those subjects were selected to have intestinal biopsies performed. There was a significant correlation between levels of platelet and jejunal mucosal TS PST activity (rs = 0.574, p < 0.030), but there was not a significant correlation between levels of TL PST activity in the two tissues (rs = 0.265, p = 0.368). There was also a significant correlation between the trait of TS PST thermal stability in the two tissues (rs = 0.828, p < 0.0001). These observations suggest that inherited variations in TS PST activity and thermal stability in an easily obtained tissue, the platelet, might be used to predict individual differences of those properties of the enzyme in the human small intestine, an organ that plays an important role in drug metabolism.

Sulfate and methyldopa metabolism: metabolite patterns and platelet phenol sulfotransferase activity.

Sulfate conjugation catalyzed by phenol sulfotransferase (PST) is the major metabolic pathway for methyldopa. Methyldopa is also O‐methylated in a reaction catalyzed by catechol‐O‐methyltransferase (COMT). Our studies were performed to determine whether sodium sulfate alters methyldopa metabolism. Methyldopa powder, 3.5 mg/kg, was taken with and without sodium sulfate, 13.25 mg/kg, by 24 subjects in a randomized, crossover design. Compared with results obtained when only methyldopa was taken, sodium sulfate taken with methyldopa increased the proportion of drug excreted as methyldopa sulfate expressed as the percentage of all urinary metabolites (66.0% ± 5.3% and 50.1% ± 7.5%; X̄ ± SD). The percentage of free methyldopa excreted also decreased (17.1% ± 3.7% and 27.3% ± 5.5%). Platelet PST and red blood cell COMT activities were measured in blood samples from these subjects. When sodium sulfate was taken with methyldopa, there was a significant correlation between platelet PST activities and percentages of metabolites excreted as methyldopa sulfate (r = 0.545; P < 0.01). This correlation was not significant when methyldopa was taken alone (r = −0.340; P > 0.10). There was a significant correlation between red blood cell COMT activities and the proportion of urinary metabolites excreted as 3‐O‐methyl‐α‐methyldopa when methyldopa was taken alone (r = 0.532; P < 0.01) but not when it was taken with sodium sulfate (r = 0.153; P > 0.20). Our data support the conclusion that variation in sulfate availability may be one factor responsible for individual differences in the metabolism of clinically used doses of methyldopa. Platelet PST activity correlates with individual variations in the sulfate conjugation of methyldopa taken by mouth with sodium sulfate.

A schizophrenic patient's attempt to resume dialysis following renal transplantation

Received January 29, 1997; revised May 21, 1997; accepted June 5, 1997. From the Department of Psychiatry and Psychology, Mayo Clinic, Rochester, Minnesota. Address reprint requests to Dr. Krahn, Department of Psychiatry and Psychology, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. Copyright 1998 The Academy of Psychosomatic Medicine. Though limited to case reports, information on patients with schizophrenia undergoing solid-organ and bone-marrow transplantation has generally been positive. Patients with psychotic disorders have been considered appropriate candidates for transplantation but are expected to have special needs. Nonetheless, the special needs of these patients are unclear and will most likely depend upon the individual circumstances of each patient and their previous psychiatric history. In a 1993 survey of transplant programs, only 33% of heart, 15% of liver, and 6% of renal transplant programs indicated that controlled schizophrenia was an absolute contraindication to transplant. We report a patient with chronic paranoid schizophrenia, who was selected for and underwent cadaver renal transplant, and required special intervention in the posttransplant period.